Multiple laminar flow-based particle and cellular identification

ABSTRACT

An apparatus and method to identify at least one component from a plurality of components in a fluid mixture, includes a first input channel containing the fluid mixture of components; at least one buffer input channel, into which at least one additional flow of buffer solution is introduced; a plurality of regions disposed at the other end of the apparatus, which are adapted to receive outputs of at least one selected component of the plurality of components, the selected component which is selectively removed from the first flow to one of the regions; a waste channel through which unselected components are removed from the first flow; a plurality of pumps connected to at least one reservoir, to control flow rates of the first flow and the additional flow(s); and a computer which controls a selection of one of the plurality of components from the fluid mixture.

CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY CLAIMS

The present invention is a continuation-in-part of Daniel M. Mueth etal., U.S. patent application Ser. No. 10/867,328, filed Jun. 13, 2004,entitled “Multiple Laminar Flow-Based Rate Zonal Or Isopycnic SeparationWith Holographic Optical Trapping Of Blood Cells And Other StaticComponents”, commonly assigned herewith, the contents of which areincorporated by reference herein, with priority claimed for all commonlydisclosed subject matter (the “first related application”).

The present invention is related to Jessica Shireman et al., U.S.Provisional Patent Application Ser. No. 60/571,141, filed May 14, 2004,entitled “System and Method of Sorting Blood Cells Using HolographicLaser Steering”, commonly assigned herewith, the contents of which areincorporated by reference herein, with priority claimed for all commonlydisclosed subject matter (the “second related application”).

The present invention is related to and a conversion to a full utilityapplication of Daniel M. Mueth, U.S. Patent Application Ser. No.60/499,957, filed Sep. 4, 2003, entitled “Passive Fluidic Sorter”,commonly assigned herewith, the contents of which are incorporated byreference herein, with priority claimed for all commonly disclosedsubject matter (the “third related application”).

The present invention is related to and a conversion to a full utilityapplication of Daniel M. Mueth, U.S. Patent Application Ser. No.60/511,458, filed Oct. 15, 2003, entitled “Passive Fluidic Sorter”,commonly assigned herewith, the contents of which are incorporated byreference herein, with priority claimed for all commonly disclosedsubject matter (the “fourth related application”).

The present invention is related to Lewis Gruber et al., U.S. patentapplication Ser. No. 10/630,904, filed Jul. 31, 2003, entitled “Systemand Method of Sorting Materials Using Holographic Laser Steering”,commonly assigned herewith, the contents of which are incorporated byreference herein, with priority claimed for all commonly disclosedsubject matter (the “fifth related application”).

FIELD OF THE INVENTION

The present invention relates generally to techniques and systems forseparation of particulate or cellular materials such as blood, semen andother particles or cells into their various components and fractions,using multiple laminar flows which further may be coupled with lasersteering such as holographic optical tapping and manipulation.

There are several categories of blood cells. Erythrocyte or red bloodcell (RBC) counts are for women 4.8 million cells/μl and men 5.4 millioncells/μl RBCs make up 93% of the solid element in blood and about 42% ofblood volume. Platelets are 2 μm-3 μm in size. They represent 7% of thesolid elements in blood and about 3% of the blood volume, correspondingto about 1.5 to 4×10¹¹ cells per liter. There are 5 general types ofwhite blood cells (WBCs) or leukocytes accounting for about 1.5 to 4×10⁹cells per liter. The WBCs comprise: 50-70% Neutrophils (12-15 μm insize); 2-4% Eosinophils (12-15 μm in size); 0.5-1% Basophils (9-10 μm insize); 20-40% Lymphocytes (25% B-cells and 75% T-cells) (8-10 μm insize); and 3-8% Monocytes (16-20 μm in size). They comprise 0.16% of thesolid elements in the blood, and approximately 0.1% of the blood volumecorresponding to around 4 to 12×10⁹ per liter. A subject with aninfection might have a WBC count as high as 25×10⁹ per liter.

Platelets are the smallest cells in the blood and are important forreleasing proteins into the blood that are involved in clotting.Patients with immune diseases that cause lower counts (such as cancer,leukemia and other chemotherapy patients) sometimes need platelettransfusions to prevent their counts from becoming too low. The plateletcount in adults is normally between 140,000-440,000 cells/μl, and thisnumber should not fall below 50,000 cells/μL because platelets play anintegral role in blood clotting.

Blood separation techniques have traditionally employed discretecentrifugation processes. More particularly, a certain volume of bloodis removed from a donor at a particular time. That volume of blood isthen subjected to different levels of centrifugation to providecorresponding blood fractions for blood components such as plasma,platelets, red blood cells, and white blood cells. This process isdiscrete, rather than continuous, such that if more blood from the donoris to be processed, another volume is removed from the donor, and theprocess is repeated.

The steps in platelet collection are: collection of blood from donor:addition of anticoagulant; separation via centrifugation; return of redcells, leukocytes and plasma to the donor. A collection normallycontains about 200-400 ml of plasma, which is reduced to avoidincompatibility. This collection normally contains about 8 to 8.5×10¹⁰platelets. A donor normally gives approximately 10% of his/her plateletswith no loss in clotting ability, although a larger number of plateletscould be separated from the blood. These platelets must be used withinfive days of collection.

Plateletpheresis, called apheresis, is a state of the art process bywhich platelets are separated [Haemonetics Component Collection System(CCS) and Multi Component System (Multi)(Haemonetics, Braintree,Mass.)]. This automated machine separates platelets from blood over aperiod of 13 to 2 hours (assuming 10% donation). This process is fasterthan traditional approaches and is completely automated and can be usedfor single or double platelet doses. Nevertheless, the process is slowrelative to the patience of donors and is capable of improvement for thepurity of the separated platelet fraction.

Other procedures are also time consuming, often taking several hours,particularly when unused blood fractions are to be returned to thedonor. For example, platelet donation make take several hours, as wholeblood is removed from the donor, fractionated through centrifugation toobtain the platelets, and the remaining blood components are theninjected back into the donor. This centrifugation process is alsocomparatively harsh, also can result in damage to a proportion of theharvested cells, effectively reducing the usable yield of the bloodfractions.

Other types of separations are also either time consuming or cannotprocess large volumes of material in a timely fashion. For example,sperm sorting, in which viable and motile sperm are isolated fromnon-viable or non-motile sperm, is often a time-consuming task, withsevere volume restrictions.

As discussed below in greater detail in describing the presentinvention, manipulations of particles, such as that described in thesecond and fifth related applications, may also be part of a novelseparation technique. One conventional technique in manipulatingmicroscopic objects is optical trapping. An accepted description of theeffect of optical trapping is that tightly focused light, such as lightfocused by a high numerical aperture microscope lens, has a steepintensity gradient. Optical traps use the gradient forces of a beam oflight to trap a particle based on its dielectric constant.

To minimize its energy, a particle having a dielectric constant higherthan the surrounding medium will move to the region of an optical trapwhere the electric field is the highest. Particles with at least aslight dielectric constant differential with their surroundings aresensitive to this gradient and are either attracted to or repelled fromthe point of highest light intensity, that is, to or from the lightbeam's focal point. In constructing an optical trap, optical gradientforces from a single beam of light are employed to manipulate theposition of a dielectric particle immersed in a fluid medium with arefractive index smaller than that of the particle, but reflecting,absorbing and low dielectric constant particles may also be manipulated.

The optical gradient force in an optical trap competes with radiationpressure which tends to displace the trapped particle along the beamaxis. An optical trap may be placed anywhere within the focal volume ofan objective lens by appropriately selecting the input beam'spropagation direction and degree of collimation. A collimated beamentering the back aperture of an objective lens comes to a focus in thecenter of the lens' focal plane while another beam entering at an anglecomes to a focus off-center. A slightly diverging beam focusesdownstream of the focal plane while a converging beam focuses upstream.Multiple beams entering the input pupil of the lens simultaneously eachform an optical trap in the focal volume at a location determined by itsangle of incidence. The holographic optical trapping technique uses aphase modifying diffractive optical element to impose the phase patternfor multiple beams onto the wavefront of a single input beam, therebytransforming the single beam into multiple traps.

Phase modulation of an input beam is preferred for creating opticaltraps because trapping relies on the intensities of beams and not ontheir relative phases. Amplitude modulations may divert light away fromtraps and diminish their effectiveness.

When a particle is optically trapped, optical gradient forces exerted bythe trap exceed other radiation pressures arising from scattering andabsorption. For a Gaussian TEM₀₀ input laser beam, this generally meansthat the beam diameter should substantially coincide with the diameterof the entrance pupil. A preferred minimum numerical aperture to form atrap is about 0.9 to about 1.0.

One difficulty in implementing optical trapping technology is that eachtrap to be generated generally requires its own focused beam of light.Many systems of interest require multiple optical traps, and severalmethods have been developed to achieve multiple trap configurations. Oneexisting method uses a single light beam that is redirected betweenmultiple trap locations to “time-share” the beam between various traps.However, as the number of traps increases, the intervals during whicheach trap is in its “off” state may become long for particles to diffuseaway from the trap location before the trap is re-energized. All theseconcerns have limited implementations of this method to less than about10 traps per system.

Another traditional method of creating multi-trap systems relies onsimultaneously passing multiple beams of light through a single highnumerical aperture lens. This is done by either using multiple lasers orby using one or more beam splitters in the beam of a single laser. Oneproblem with this technique is that, as the number of traps increases,the optical system becomes progressively more and more complex. Becauseof these problems, the known implementations of this method are limitedto less than about 5 traps per system.

In a third approach for achieving a multi-trap system, a diffractiveoptical element (DOE) (e.g., a phase shifting hologram utilizing eithera transmission or a reflection geometry) is used to alter a single laserbeam's wavefront. This invention is disclosed in U.S. Pat. No. 6,055,106to Grier et al. The wavefront is altered so that the downstream laserbeam essentially becomes a large number of individual laser beams withrelative positions and directions of travel fixed by the exact nature ofthe diffractive optical element. In effect, the Fourier transform of theDOE produces a set of intensity peaks each of which act as an individualtrap or “tweezer.”

Some implementations of the third approach have used a fixedtransmission hologram to create between 16 and 400 individual trappingcenters.

A fixed hologram has been used to demonstrate the principle ofholographic optical trapping but using a liquid crystal grating as thehologram permitted ‘manufacture’ of a separate hologram for each newdistribution of traps. The spatially varying phase modulation imposed onthe trapping laser by the liquid crystal grating may be easilycontrolled in real time by a computer, thus permitting a variety ofdynamic manipulations.

Other types of traps that may be used to optically trap particlesinclude, but are not limited to, optical vortices, optical bottles,optical rotators and light cages. An optical vortex produces a gradientsurrounding an area of zero electric field which is useful to manipulateparticles with dielectric constants lower than the surrounding medium orwhich are reflective, or other types of particles which are repelled byan optical trap. To minimize its energy, such a particle will move tothe region where the electric field is the lowest, namely the zeroelectric field area at the focal point of an appropriately shaped laserbeam. The optical vortex provides an area of zero electric field muchlike the hole in a doughnut (toroid). The optical gradient is radialwith the highest electric field at the circumference of the doughnut.The optical vortex detains a small particle within the hole of thedoughnut. The detention is accomplished by slipping the vortex over thesmall particle along the line of zero electric field.

The optical bottle differs from an optical vortex in that it has a zeroelectric field only at the focus and a non-zero electric field in allother directions surrounding the focus, at an end of the vortex. Anoptical bottle may be useful in trapping atoms and nanoclusters whichmay be too small or too absorptive to trap with an optical vortex oroptical tweezers. (See J. Arlt and M. J. Padgett. “Generation of a beamwith a dark focus surrounded by regions of higher intensity: The opticalbottle beam,” Opt. Lett. 25, 191-193, 2000.)

The light cage (U.S. Pat. No. 5,939,716) is loosely, a macroscopiccousin of the optical vortex. A light cage forms a time-averaged ring ofoptical traps to surround a particle too large or reflective to betrapped with dielectric constants lower than the surrounding medium.

When the laser beam is directed through or reflected from the phasepatterning optical element, the phase patterning optical elementproduces a plurality of beamlets having an altered phase profile.Depending on the number and type of optical traps desired, thealteration may include diffraction, wavefront shaping, phase shifting,steering, diverging and converging. Based upon the phase profile chosen,the phase patterning optical element may be used to generate opticaltraps in the form of optical traps, optical vortices, optical bottles,optical rotators, light cages, and combinations of two or more of theseforms.

Researchers have sought indirect methods for manipulating cells, such astagging the cells with diamond micro-particles and then tweezing thediamond particles. Cell manipulations have included cell orientation formicroscopic analysis as well as stretching cells. Tissue cells have alsobeen arranged with tweezers in vitro in the same spatial distribution asin vivo.

In addition to the cells themselves, optical tweezers have been used tomanipulate cellular organelles, such as vesicles transported alongmicrotubules, chromosomes, or globular DNA. Objects have also beeninserted into cells using optical tweezers.

Accordingly, as an example of new types of sorting using laser steeredoptical traps, a method of cell sorting using a technique which isolatesvaluable cells from other cells, tissues, and contaminants is needed.Further, a way of achieving a unique contribution of optical trapping tothe major industrial needs of blood cell sorting and purification isrequired. Still further, there is a need to separate sperm cells in theanimal husbandry market.

As a consequence, a need remains for a separation technique andapparatus which is continuous, has high throughput, provides timesaving, and which causes negligible or minimal damage to the variouscomponents for separation. In addition, such techniques should havefurther applicability to biological or medical areas, such as forseparations of blood, sperm, other cellular materials, as well as viral,cell organelle, globular structures, colloidal suspensions, and otherbiological materials.

SUMMARY OF THE INVENTION

The exemplary embodiments of the present invention provide forseparating components in a mixture, such as separating the various bloodcomponents of whole blood into corresponding fractions, such as aplatelet fraction, a red blood cell fraction, a white blood cellfraction, and a plasma fraction. The various embodiments of the presentinvention provide separation of components on a continuous basis, suchas within a continuous, closed system, without the potential damage andcontamination of prior art methods, particularly for fractionation ofblood components. The continuous process of the present invention alsoprovides significant time savings and higher throughput for bloodfractionation. In addition, the various embodiments may also includeadditional means for separating and manipulating the components,particularly holographic optical manipulation and separation. Thevarious embodiments may also be applied to separations of other types ofcellular and biological materials, such as sperm, viruses, bacteria,cell debris, cell organelles, globular structures, colloidalsuspensions, cellular debris, and other biological materials.

As used herein, “Particle” refers to a biological or other chemicalmaterial including, but not limited to, oligonucleotides,polynucleotides, chemical compounds, proteins, lipids, polysaccharides,ligands, cells, antibodies, antigens, cellular organelles, lipids,blastomeres, aggregations of cells, microorganisms, peptides, cDNA, RNAand the like.

An exemplary method of separating blood into components includesproviding a first flow having a plurality of blood components; providinga second flow; contacting the first flow with the second flow to providea first separation region; and differentially sedimenting a first bloodcellular component of the plurality of blood components into the secondflow while concurrently maintaining a second blood cellular component ofthe plurality of blood components in the first flow. The second flowhaving the first blood cellular component is then differentially removedfrom the first flow having the second blood cellular component.

The various sedimentation steps of the present invention may be ratezonal or isopycnic. In addition, the first flow and the second flow aresubstantially non-turbulent, and may also be substantially laminar.

In a selected embodiment, the first blood cellular component is aplurality of red blood cells and a plurality of white blood cells, andthe second blood cellular component is a plurality of platelets. For thefirst blood cellular component, the plurality of white blood cells maybe holographically separated (through laser steering) from the pluralityof red blood cells. Other holographic manipulations of the presentinvention include holographically removing a plurality of contaminantsfrom the first flow, holographically separating biological debris fromthe first flow, and holographically separating a plurality of secondblood cellular components from the first flow.

Additional separation stages may also be included, with the exemplarymethod providing a third flow; contacting the first flow with the thirdflow to provide a second separation region; and differentiallysedimenting the second blood cellular component of the plurality ofblood components to sediment into the third flow while concurrentlymaintaining a third blood component of the plurality of blood componentsin the first flow. In selected embodiments, the second blood cellularcomponent is a plurality of platelets and wherein the third bloodcomponent is plasma.

A plurality of separation stages may also be combined to form morecomplicated structures having multiple separation stages, connected inseries, connected in parallel, or in combinations of both.

A second exemplary method of separating a fluid mixture intoconstituent, non-motile components, in accordance with the presentinvention, includes: providing a substantially laminar first flow havingthe fluid mixture, the fluid mixture having a plurality of components,the plurality of components having a corresponding plurality ofsedimentation rates; providing a substantially laminar second flow;contacting the first flow with the second flow to provide a firstseparation region, the first flow and the second flow having asubstantially non-turbulent interface within the separation region;differentially sedimenting from the first flow a first component of theplurality of components into the second flow to form an enriched secondflow and a depleted first flow, while concurrently maintaining a secondcomponent of the plurality of components in the first flow, the firstcomponent having a first sedimentation rate of the plurality ofsedimentation rates and the second component having a secondsedimentation rate of the plurality of sedimentation rates, wherein thefirst sedimentation rate is comparatively greater than the secondsedimentation rate; differentially removing the enriched second flowfrom the depleted first flow; and holographically manipulating thesecond component in the depleted first flow.

The second exemplary method may also include additional separationstages, such as a holographic separation, including: providing a thirdflow; contacting the depleted first flow with the third flow to providea second separation region; and holographically trapping the secondcomponent and moving the second component from the depleted first flowinto the third flow while concurrently maintaining a third component ofthe plurality of components in the depleted first flow.

An exemplary apparatus embodiment of the invention for separating afluid mixture into constituent, non-motile components includes: a firstsorting channel having a first inlet for a first flow and a second inletfor a second flow; the first sorting channel further having a firstoutlet for the first flow and a second outlet for the second flow, thefirst sorting channel further having means to maintain the first flowand second flow substantially non-turbulent, the first sorting channeladapted to allow a first component in the first flow, of a plurality ofcomponents in the first flow, to sediment into the second flow to forman enriched second flow and a depleted first flow, while concurrentlymaintaining a second component of the plurality of components in thefirst flow; a second, optically transparent sorting channel having afirst optical inlet coupled to the first outlet for the first flow andhaving a first optical outlet, the second, optically transparent sortingchannel further having a second optical inlet for a third flow and asecond optical outlet for the third flow; and a holographic optical trapcoupled to the second, optically transparent sorting channel, theholographic optical trap adapted to generate a holographic optical trapto select and move the second component from the first flow into thethird flow. The various components which are separated, for example, maybe the various blood fractions or other biological materials, such asseparations of motile from non-motile sperm.

Another apparatus or system for separating a plurality of components ina fluid comprises: an optically transparent sorting channel having afirst inlet for a first flow and a second inlet for a second flow, theoptically transparent sorting channel further having a first outlet forthe first flow and a second outlet for the second flow; and aholographic optical trap system coupled to the optically transparentsorting channel, the holographic optical trap system adapted to generatea holographic optical trap to select and move a first component in thefirst flow, of a plurality of components in the first flow, into thesecond flow to form an enriched second flow and a depleted first flow,while a second component of the plurality of components is concurrentlymaintained in the first flow.

Another method embodiment provides for separating a plurality of cells,comprising: providing a first flow having the plurality of cells;providing a second flow; contacting the first flow with the second flowto provide a first separation region; and differentially sedimenting afirst cell of the plurality of cells into the second flow whileconcurrently maintaining a second cell of the plurality of cells in thefirst flow. The method generally also includes differentially removingthe second flow having the first cell from the first flow having thesecond cell. The method may also provide for providing a third flow;contacting the first flow with the third flow to provide a secondseparation region; and differentially sedimenting the second cell of theplurality of cells into the third flow while concurrently maintaining athird cell of the plurality of cells in the first flow. In addition, aplurality of second cells may be holographically separated from thefirst flow, and a plurality of contaminants or biological debris may beholographically removed from the first flow.

In another embodiment consistent with the present invention, opticaltrapping for laser steering), which is a technology which has been usedas a tool for manipulating microscopic objects, is used. An accepteddescription of the effect is that tightly focused light, such as lightfocused by a high numerical aperture microscope lens, has a steepintensity gradient. Optical traps use the gradient forces of a beam oflight to trap a particles based on its dielectric constant To minimizeits energy, a particle having a dielectric constant higher than thesurrounding medium will move to the region of an optical trap where theelectric field is the highest.

Optical trapping of the present invention is used to address cellsorting and purification (e.g., from contaminants such as viruses andbacteria) in several ways. For example, the forces exerted by opticaltraps on a material are sensitive to the exact distribution of thedielectric constant in that material—the optical force therefore dependson the composition and shape of the object.

Further, other forces on the object are sensitive to the hydrodynamicinteraction between the object and the surrounding fluid—control of thefluid flow probes material shape, size and such features as surfacerugosity.

Still further, localizing an object at a known position allowsadditional methods of automated interrogation such as high speed imagingand particle-specific scattering measurements.

In one embodiment consistent with the present invention, in achieving amulti-trap system, a diffractive optical element (“DOE”, i.e., a phaseshifting hologram utilizing either a transmission or a reflectiongeometry) is used to alter a single laser beam's wavefront. Thewavefront is altered so that the downstream laser beam essentiallybecomes a large number of individual laser beams with relative positionsand directions of travel fixed by the exact nature of the diffractiveoptical element.

The present invention provides optical trapping by focusing a laser beamwith a lens to create an optical trap wherein the lens has a numericalaperture less than 0.9, and preferably decreases until it is mostpreferably less than 0.1.

Sorting using holographic laser steering involves establishing classesof identification for objects to be sorted, introducing an object to besorted into a sorting area, and manipulating the object with a steeredlaser according to its identity class. The manipulation may be holding,moving, rotating, tagging or damaging the object in a way which differsbased upon its identity class. Thus, the present invention provides away of implementing a parallel approach to blood cell sorting and spermcell sorting using holographic optical trapping.

In one embodiment of the present invention, spectroscopy of a sample ofbiological material may be accomplished with an imaging illuminationsource suitable for either inelastic spectroscopy or polarized lightback scattering, the former being useful for assessing chemicalidentity, and the latter being suited for measuring dimensions ofinternal structures such as the nucleus size. Using such spectroscopicmethods, in some embodiments, cells are interrogated. The spectrum ofthose cells which had positive results (i.e., those cells which reactedwith or bonded with a label) may be obtained by using this imagingillumination.

A computer program may analyze the spectral data to identify the desiredtargets (i.e., cells bearing either an X or Y chromosome, or a suspectedcancerous, pre-cancerous and/or non-cancerous cell types, etc.), thenmay apply the information to direct the phase patterning optical element(i.e., optical traps) to segregate or contain those desired or selectedtargets (i.e., cell types). The contained cells may be identified basedon the reaction or binding of the contained cells with chemicals, or byusing the natural fluorescence of the object, or the fluorescence of asubstance associated with the object, as an identity tag or backgroundtag. Upon completion of the assay, selection may be made, via computerand/or operator, of which cells to discard and which to collect.

Manipulation of cells in general, is made safer by having multiple beamsavailable. Like a bed of nails, multiple tweezers ensure that less poweris introduced at any particular spot in the cell. This eliminates hotspots and reduces the risk of damage. Any destructive two-photonprocesses benefit greatly since the absorption is proportional to thesquare of the laser power. Just adding a second tweezer decreasestwo-photon absorption in a particular spot by a factor of four. Trappinglarge cells involves a large amount of laser power for effectivetrapping. Putting the power into a single trap may cause immediatedamage to the cell.

The manipulation of even just a single cell is greatly enhanced byutilizing holographic optical trapping, for example. A single cell maybe manipulated by a line of tweezers, which lift the cell along theperimeter on one side. The resulting rotation allows a 360 degree viewof the cell. In addition to the advantage for viewing of biologicalsamples, there also exists the ability to orient samples stably, whichhas clear benefit for studies such as scattering experiments which havea strong dependence on orientation of the sample.

Sorting with a wide field of view has many advantages such as higherthroughput. However, standard tweezing in a WFOV (wide field of view)may fail due to excessive radiation pressure. Tweezing with a wide fieldof view using holographic optical trapping may permit the ability toform exotic modes of light which greatly reduce the radiation pressureof the light beam. Vortex traps, for example, have a dark center becausethe varying phases of light cancel in the center of the trap. This darkcenter means most of the rays of light which travel down the center ofthe beam no longer exist. It is exactly these beams which harbor most ofthe radiation pressure of the light, so their removal greatly mitigatesthe difficulty in axial trapping. Other modes, e.g., donut modes, havethe same advantage.

In one embodiment consistent with the present invention, the method andsystem lends itself to a semi-automated or automated process fortracking the movement and contents of each optical trap. In oneembodiment consistent with the present invention, movement may bemonitored via an optical data stream which can be viewed, or convertedto a video signal, monitored, or analyzed by visual inspection of anoperator, spectroscopically, and/or by video monitoring. The opticaldata stream may also be processed by a photodectector to monitorintensity, or any suitable device to convert the optical data stream toa digital data stream adapted for use by a computer and program. Thecomputer program controls the selection of cells and the generation ofoptical traps.

In other embodiments consistent with the present invention, the movementof cells is tracked based on predetermined movement of each optical trapcaused by encoding the phase patterning optical element. Additionally,in some embodiments, a computer program maintains a record of each cellcontained in each optical trap.

There has thus been outlined, rather broadly, some features consistentwith the present invention in order that the detailed descriptionthereof that follows may be better understood, and in order that thepresent contribution to the art may be better appreciated. There are, ofcourse, additional features consistent with the present invention thatwill be described below and which will form the subject matter of theclaims appended hereto.

In this respect; before explaining at least one embodiment consistentwith the present invention in detail, it is to be understood that theinvention is not limited in its application to the details ofconstruction and to the arrangements of the components set forth in thefollowing description or illustrated in the drawings. Methods andapparatuses consistent with the present invention are capable of otherembodiments and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein, as well as the abstract included below, are for thepurpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe methods and apparatuses consistent with the present invention.

Numerous other advantages and features of the present invention willbecome readily apparent from the following detailed description of theinvention and the embodiments thereof, from the claims and from theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects, features and advantages of the present invention will bemore readily appreciated upon reference to the following disclosure whenconsidered in conjunction with the accompanying drawings, in which:

FIG. 1 is a lateral view of an apparatus 100 in accordance with oneembodiment consistent with the present invention.

FIG. 2 is an illustration of optical trapping for component separationin an apparatus 200.

FIG. 3 is a diagram illustrating a closed, two-stage system 300 forblood component separation in accordance with one embodiment consistentwith the present invention.

FIG. 4 schematically illustrates a holographic optical trapping systemin accordance with one embodiment consistent with the present invention.

FIG. 5 is a schematic diagram of a holographic optical trapping systemfor sorting objects in accordance with one embodiment consistent withthe present invention.

FIG. 6 (divided into FIG. 6A and FIG. 6B) is a flow diagram illustratinga method embodiment of consistent with the present invention.

FIGS. 7A and 7B are a side (lateral) view schematic diagram and a topview schematic diagram, respectively, showing a sample being introducedinto sample holder, in accordance with one embodiment consistent withthe present invention.

FIG. 8 depicts a scanning electron micrograph of a sample chamber inaccordance with one embodiment consistent with the present invention.

FIG. 9 shows an enlarged view of the working area of a sample chamber inaccordance with one embodiment consistent with the present invention.

FIG. 10 illustrates an example of lateral deflection for sorting inaccordance with one embodiment consistent with the present invention.

FIGS. 11A and 11B illustrate schematic front and side views,respectively, of the funneling traps in accordance with one embodimentconsistent with the present invention.

FIG. 12 illustrates a spinning disc-based cell sorter in accordance withone embodiment consistent with the inventions of the second and fifthrelated applications.

FIG. 13 illustrates optical peristalsis in accordance with oneembodiment consistent with the present invention.

FIG. 14 illustrates a sorting system in accordance with one embodimentconsistent with the present invention.

FIG. 15 illustrates a sorting system in accordance with one embodimentconsistent with the present invention.

FIG. 16 is a lateral view of a high-aspect ratio flat sorter inaccordance with one embodiment consistent with the present invention.

FIG. 17 is a plan view of a high-aspect ratio flat sorter in accordancewith one embodiment consistent with the present invention.

FIG. 18 is a perspective view of a three-dimensional sorting devicehaving a plurality of flat sorters in accordance with one embodimentconsistent with the present invention.

FIG. 19 is a plan view of a multi-channel sorter in accordance with oneembodiment consistent with the present invention.

FIG. 20 is a plan view of a sorter having a narrow waste flow region inaccordance with one embodiment consistent with the present invention.

FIG. 21 is a plan view of a sorter using different flow rates forvarious channels in accordance with one embodiment consistent with thepresent invention.

FIG. 22 is a plan view of a sorter having multiple selection channels inaccordance with one embodiment consistent with the present invention.

FIG. 23 is a plan view of a sorter having a constricted sorting regionin accordance with one embodiment consistent with the present invention.

FIG. 24A is a lateral view of a multi-layer laminar flow sorter inaccordance with one embodiment consistent with the present invention.

FIG. 24B is a plan view of a multi-layer laminar flow sorter inaccordance with one embodiment consistent with the present invention.

FIG. 25 illustrates the results of bovine sperm viability or motilitysorting using the various embodiments of the present invention.

FIG. 26 is a block diagram illustrating an exemplary sorting andseparation system in accordance with one embodiment consistent with thepresent invention.

FIG. 27 is a block diagram illustrating an exemplary bioreactor productpurification and separation system in accordance with one embodimentconsistent with the present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

While the present invention is susceptible of embodiment in manydifferent forms, there are shown in the drawings and will be describedherein in detail specific embodiments thereof, with the understandingthat the present disclosure is to be considered as an exemplification ofthe principles of the invention and is not intended to limit theinvention to the specific embodiments illustrated.

As indicated above, the various embodiments of the present inventionprovide for separating components in a mixture, such as separating thevarious blood components of whole blood into corresponding fractions,such as a platelet fraction, a red blood cell fraction, a white bloodcell fraction, and a plasma fraction. The various embodiments, asdescribed below, utilize one or more sorting channels, having aplurality of substantially laminar flows, allowing one or morecomponents to differentially sediment from one flow into another,thereby separating the components into corresponding flows. In addition,the various components may be sorted further using optical mechanisms,such as holographic optical trapping. The various embodiments of thepresent invention thereby provide separation of components on acontinuous basis, such as within a continuous, closed system, withoutthe potential damage and contamination of prior art methods,particularly for fractionation of blood components. The continuousprocess of the present invention also provides significant time savingsfor blood fractionation.

In addition to whole blood sorting and fractionation applications, thepresent invention is also suitable for other cell sorting applications,such as separations of cancer cells from normal or healthy cells in, forexample, bone marrow extractions. The various embodiments of the presentinvention have further applicability to other biological or medicalareas, such as for separations of cells, sperm, viruses, bacteria,cellular organelles or subparts, globular structures, colloidalsuspensions, lipids and lipid globules, gels, immiscible particles,blastomeres, aggregations of cells, microorganisms, and other biologicalmaterials. For example, the component separation in accordance with thepresent invention may include cell “washing”, in which contaminants(such as bacteria) are removed from cellular suspensions, which may beparticularly useful in medical and food industry applications.Significantly, prior art flow-based techniques have not recognized anyapplicability to sorting or separation of non-motile cellular componentsusing variable sedimentation rates and optical manipulation.

While discussion below focuses on the sorting of blood components tocreate different blood fractions, the apparatus, methods and systems ofthe present invention may be extended to other types of particulate,biological or cellular matter, which are capable of sedimenting orcreaming within a fluid flow, or which are capable of being manipulatedoptically between different fluid flows. For example, the methodology ofthe present invention could be utilized to separate non-motile ornon-viable sperm cells from viable cells, by allowing the non-motilecells to sediment from a first flow into a second flow while retainingthe motile cells in the first flow or allowing the motile cells to moveto a third flow. Other sorts of cell separation may also be performed,such as separating islet cells from other types of pancreatic cells, orotherwise separating islet cell clusters of different sizes, througheither or both flow separation or optical tweezing (trapping). Viruses,proteins and other large molecules having different sedimentation ratesmay also be separated with the present invention. The holographicoptical trapping utilized with the various separation stages may also beparticularly useful in these other types of cell or particleseparations.

The present invention has other medical applications as well. Forexample, the various laminar flows discussed below may be utilized aspart of a kidney dialysis process, in which whole blood is cleansed ofwaste products and returned to the patient. As a consequence, inaddition to particle separations based upon relative density, forexample, the present invention may be utilized for separations basedupon diffusion, motility, and other types of gradients.

For example, the present invention may be utilized to move a speciesfrom one solution to another solution where separation by filtering orcentrifugation is not practical or desirable. In addition to theapplications discussed above, additional applications include isolatingcolloids of a given size from colloids of other sizes (for research orcommercial applications), and washing particles such as cells, eggcells, etc. (effectively replacing the medium in which they arecontained and removing contaminants), or washing particles such asnanotubes from a solution of salts and surfactants with a different saltconcentration or without surfactants, for example.

The action of separating species may rely on a number of physicalproperties of the objects including self-motility, self-diffusivity,free-fall velocity, or action under an external force, such as anelectromagnetic field or a holographic optical trap. The propertieswhich may be sorted upon include cell motility, cell viability, objectsize, object mass, object density, the tendency of particles to attractor repel one another or other objects in the flow, object charge, objectsurface chemistry, and the tendency of certain molecules to adhere tothe object.

While the present invention is discussed in detail with respect to theapparatus 100, 200 and 300, it should be understood that this discussionapplies equally to the various other embodiments illustrated in FIGS.13-24 and 26-27.

FIG. 1 is an illustration of a lateral view of an apparatus 100 inaccordance with the present invention. As illustrated in FIG. 1, thesorting apparatus 100 includes a sorting channel 110, a plurality ofinlets 120 and a plurality of outlets 130. A corresponding fluid flow,such as illustrated flows W, X, Y and Z, enters one of the inlets 120and flows, substantially non-turbulently or otherwise as a laminar flow,across the sorting channel (or sorting region) 110, and out through acorresponding outlet 130, as illustrated.

The apparatus 100 (and 200, below) may be constructed of a plurality ofmaterials, integrally or as discrete components, using a wide variety ofmaterials, such as metals, ceramics, glass, and plastics. Variousmaterials and fabrication methods are discussed in the third and fourthrelated applications, and include, for example, use of various polymerswhich cure under UV exposure. Other details are also provided in thethird and fourth related applications, such as the use and selection ofdifferent types of pumps, such as syringe pumping, peristaltic pumping,gravity-driven pumping, and various combinations of pumping actions.

In selected embodiments, when coupled with holographic trapping or otherform of optical tweezing, the apparatus 100 (or 200) is transparent tothe selected wavelength of the holographic generator, such as opticallytransparent when the holographic generator utilizes visible wavelengths.Depending upon the selected application, the apparatus 100 (200) shouldalso be sterile and may also have a controlled temperature. The variousfluid flows may be fed into the inlets 120 through a wide variety ofmeans known to those of kill in the art and are within the scope of thepresent invention, including use of peristaltic pumps or a gravity feed,for example, and such means may also be utilized to control the flowrates of the various flows W, X, Y and Z. When peristaltic pumps areutilized, to maintain a constant flow rate and pressure, bubble-trapsmay be incorporated at the inlets 120 of the apparatus 100 (or 200).

The various fluids utilized in the separation flows may be diluted orconcentrated, to increase or decrease the volume of one of thesolutions, or to impact the concentration of some dissolved or suspendedmaterial, or to impact physical properties of the solution such as itsviscosity, temperature, or density. Examples for the apparatus 100, whenused for blood sorting, include: (a) dilution of the blood to reduceclogging or hydrodynamic interaction between blood cells, (b) extensionof the volume of the blood or a blood fraction, (c) modification of thedensity of the blood, a blood fraction, or another solution whichimpacts the flow properties or separation behavior, (d) extension of thevolume of a solution to maintain in increase fluid volume, especially incircumstances when fluid volume is being removed from the system. Asdiscussed in greater detail below, various chemical attractants andrepellants may be added to the fluids, which may also be at differenttemperatures and viscosity levels, to improve sperm sorting.

The various fluids utilized in the separation flows also may be“activated”, such that some process is activated within the solution bysome external influence or mixing with an external solution. Examples ofexternal influences include: (a) applying an electric field, (b)applying a magnetic field, (c) exposing to light, (d) modifying thetemperature, (e) introducing a chemical, (f) introducing a biologicalmaterial, (g) shearing the solution, and (h) vibrating the solution.Examples of the activation which is caused by the external solutioninclude: (a) alignment of particles, molecules, or cells, (b)polarization of one or more components of the solution, (c)cross-linking, (d) initiation or termination of chemical reaction, (e)initiation or termination of a biological response, (f) changing thetype or rate of a chemical, physical, or biological response, or (g)causing a response or separation which depends upon the character of theparticular component which is responding. Examples for the apparatus100, when used for blood sorting, include: (a) addition of an agent toreduce clotting; (b) addition of agents to preserve viability or healthof the blood solution or its components; (c) addition of agents whichmay augment the sorting process, such as by binding or collecting nearcertain components, thereby influencing one or more of their physicalproperties, including the addition of beads or other particles orpolymers which may adhere to one or more species, and also including theintroduction of salts or other materials which may influence theelectrostatic interaction of materials or the hydrodynamic size orcharacter of the materials; (d) addition of agents which may influencethe flow properties, such as by changing the density, viscosity, surfacetension, or other parameters; (e) addition of agents to enhance orsuppress the aggregation of certain materials; and (f) addition ofagents to enhance or suppress the adherence of certain materials toother materials or parts of the flow device.

In accordance with the invention, one of the fluid flows, such as theillustrated flow W, contains a plurality of components A, B, C and E.For example, when the fluid is whole blood, these components may be redblood cells (“RBC”), white blood cells (“WBC”), platelets, cellulardebris and contaminants, all in plasma. Typically, many of the pluralityof components have different sedimentation rates, typically measuredusing a Svedberg coefficient. For example, RBCs have a comparativelygreater sedimentation rate than platelets, and will be expected tosediment faster on a passive basis, such as due to gravitational orbuoyant forces, without the intervention of other, active mechanisms,such as centrifugation. As the various flaws W, X, Y and Z flow throughthe sorting region 110, based upon different sedimentation rates, theplurality of components (such as cells or other particles) willsediment, moving from one flow to another. As illustrated, component Ahaving the comparatively greatest sedimentation rate is illustrated ashaving moved from flow W to the lowest flow Z, component B having thecomparatively next highest sedimentation rate is illustrated as havingmoved from flow W to the flow Y (above Z), component C having acomparatively smaller sedimentation rate is illustrated as having movedfrom flow W to the flow X (above Y), while component E having thecomparatively smallest sedimentation rate, is illustrated as havingremained in flow W (above Y). Using these different sedimentationproperties, each of these components may be separated into acorresponding flow, and isolated from each other as each flow exitsthrough its corresponding outlet 130. As each flow W, X, Y and Z exitsthrough its corresponding outlet 130, that flow is differentiallyremoved from the other flows, i.e., the flow is removed while the otherflow remains intact or is otherwise separately removed from theremaining flows. In addition, this differential removal may beconcurrent, namely, all flows removed concurrently or continuously.

Continuing to refer to FIG. 1, using whole blood with an anticoagulant(such as sodium citrate or heparin) as the fluid flow W, for example,the various blood fractions may be separated from each other, with redblood cells sedimenting fastest and represented by component A (e.g.,4.59 μm/s), white blood cells sedimenting at a slightly lower rate andrepresented by component B (e.g., 2.28 μm/s), platelets sedimenting at acomparatively slower rate and represented by component C (e.g., 0.055μm/s), and plasma continuing to comprise flow W and represented bycomponent E. Each blood fraction may then be removed through acorresponding outlet 130.

Not separately illustrated in FIG. 1, due to buoyant forces and relativedensity considerations, there may be particles or components in one ormore of the fluid flows which will flow up to a higher flow (e.g.,creaming). For example, less dense particles entering through flow X mayrise into flow W, and exit with flow W through a corresponding outlet130.

Illustrated in lateral view, the sorting channel (or sorting region) 110of apparatus 11 has a varied length “L” parallel to the direction offlow, a depth “D” perpendicular to the direction of flow, and a width“WW”, illustrated as extending into the page (and designated WW to avoidconfusion with the W flow). These various dimensions may be selectedbased on a plurality of factors, particularly the flow rates and thesedimentation rates of the components of interest. For example, for aselected flow rate, the total length of the sorting channel should belong enough to differentially remove the component having thecomparatively slowest sedimentation rate, illustrated as component C inFIG. 2, with shorter lengths corresponding to other flows for separationof components having faster sedimentation rates, as illustrated for flowZ having component A and flow Y having component B.

The present invention is further distinguished from the prior art byhaving considerably more latitude or tolerance for aspect ratios, whilenonetheless maintaining a substantially laminar flow. The aspect ratioof length to width, for example, may vary from about 5 (or more) to 1(5:1), with the length being greater than the width, to about 1 (orless) to 2 (the length being smaller than the width). A preferred lengthto width aspect ratio is about 2:1, and may vary from 3:1 to 1:2.

Flow rates may also vary between the plurality of flows utilized inapparatus 100. For example, higher flow rates in the lower level flows(such as Y and Z) may tend to compress the flows W and X, resulting in ashorter distance that certain components must traverse to sediment intothe flows Y and Z.

In addition, the sedimentation of components through the various flowsof the apparatus 100 is typically rate zonal, that is, based upon bothrelative density and size of the components to be separated, as well asthe material's shape and electrostatic properties. Under otherconditions, however, such as slower flow rates, thinner flow depths,and/or longer sorting channels 100, the sedimentation may also beisopycnic, that is, based only upon relative density of the components.

When isopycnic separation is desired, the various fluids comprising theflows W, X, Y and Z may be selected and adjusted to create desireddensity gradients to match the component densities for the selectedseparations. For example, the various fluids comprising the flows W, X,Y and Z may be selected and adjusted to each have a different,increasing or decreasing density, creating a stepped density gradient,with various particles sedimenting to the appropriate step. In addition,through use of a sufficient number of fluid flow layers, the densitygradient will effectively become continuous, with a correspondingability for fine-grained separation.

The various fluids comprising the generally laminar flows, such as flowsW, X, Y and Z may also be selected based on suitable criteria for theparticular desired component separation. For example, for bloodseparation, the various flows may be comprised of whole blood, such asfor flow W, and plasma or buffering solutions for the remaining flows.The various fluids may also be preprocessed prior to entry through theinlets 120, such as through dilution, addition of other components suchas additives (such as anticoagulants, flocculants, or binding agents),viscosity or other flow property manipulation, or preprocessed throughother separation techniques. Also for example, whole blood may bepreprocessed to initially remove some red blood cells or to add ananticoagulant such as sodium citrate.

While illustrated with four flows or channels, it should be understoodthat the apparatus 100 (or 200, below) may be implemented with anynumber of flows and corresponding fluid inlets 120 and outlets 130. Onelimitation to the number of fluid flows is based on the ability tomaintain each flow in a substantially laminar or non-turbulent manner,such that each interface between flows is substantially non-turbulent,to minimize any unwanted mixing of flows. In addition, there also may berelative density considerations for the fluids comprising the flowswhich could also result in limiting the number of flows utilized in agiven stage of separation.

The various apparatus 100 (or 200, below) may be further coupled toadditional apparatus 100 (200, below), in parallel for higherthroughput, and in series for additional separation stages, such as forincreased purity levels. In addition, the various apparatus 100 may alsobe combined with non-sedimentation separations, or be coupled in serieswith subsequent separations using non-sedimentation mechanisms, withadditional separation of components between flows accomplished, forexample, using optical forces such as holographic optical trapping ofthe fifth related application, incorporated herein by reference. Thesevarious apparatus 100, 200 or 300, moreover, may have differentdimensions and different numbers of channels or flows.

FIG. 2 (and also FIG. 13) is a general illustration of using suchoptical forces created by holographic or optical trapping for additionalcomponent separation in an apparatus 200. Creation and manipulation ofthe plurality of holographic optical traps 210 is explained in greaterdetail below with reference to FIGS. 4 and 5, with apparatus 200 formingthe sample 506 of FIG. 5. Two flows W and X are illustrated in FIG. 2,with flow W initially having two components A and B. Holographic opticaltraps 210 (illustrated as conic sections in FIG. 2) are then utilized tocapture “A” components, and move them into flow X. Such optical trappingis particularly useful for increased purification of a particularfraction, particularly for fractions having insufficient differentiationbased on sedimentation rates. Such optical trapping is also particularlyuseful for removal of undesirable components, such as cellular debrisand other impurities. In addition, where mixing or remixing ofcomponents may have occurred during the rate zonal laminar flowseparations discussed above, the optical trapping may be particularlyaccurate in removing undesired components. For example, a comparativelysmall portion of white blood cells may not have sedimented fast enough,resulting in some white blood cell contamination of a platelet fraction.Optical trapping may be utilized to select and the white blood cellsinto a separate flow, increasing the purity of the platelet fraction.

For blood sorting applications, it should be understood that plateletsand RBC optically manipulate (or “tweeze”) better than white bloodcells. Using lower numerical apertures in the system 500 (discussedbelow), however, significantly improves optical manipulation of whiteblood cells.

When implemented in conjunction with optical traps, the apparatus 100,200 or 300 should be embodied utilizing an optically transparentmaterial, for the selected optical wavelength. When the holographictraps are implemented at other wavelengths, other correspondinglytransparent materials may be utilized which are suitable for theselected wavelength. The apparatus 100, 200 or 300 is then implementedand placed in the location of the sample 506 illustrated in FIG. 5, withthe system 500 utilized to perform the holographic optical trapping asone of or as part of a separation stage of the present invention.

FIG. 3 is a diagram illustrating a closed, two-stage system 300 forblood or other component separation in accordance with the presentinvention. In a first stage 305, blood components from a selected donorflow through inlet 315 to form a first flow, and plasma is returned (orprimed on initial start up) through inlet 320 to form a second flow. Thefirst and second flows are non-turbulent and otherwise laminar flows,and make non-turbulent contact with each other in first separationregion 325, forming a non-turbulent interface region between the twoflows. In the first separation region 325, both red blood cells andwhite blood cells sediment from the first flow into the second flow, andare collected in reservoir 330 for other uses (such as medical uses forpacked cells) or for return to the selected donor. As indicated above,both the length of the first separation region 325 and the flow rate ofthe first flow are predetermined such that both red blood cells andwhite blood cells have sufficient time to passively sediment into thesecond flow, under gravitational and buoyant forces.

Continuing to refer to FIG. 3, the first flow, now substantiallydepleted of both red blood cells and white blood cells, flowsnon-turbulently on a continuous path into a second separation stage 310.In the second separation stage 310, the first flow enters a secondseparation region 340 with a third flow from inlet 335. The third flowis also comprised of plasma from the selected donor in the exemplaryembodiment. In the second separation region 340, the platelets remainingin flow one passively sediment into flow three, and are collected withthe plasma of flow three in reservoir 345 for medical use, for example.The further depleted flow one is then recirculated from outlet 350 backto inlets 320 and 335, to form the first and third flows, respectively.As indicated above, the system 300 may be primed with donor plasma atsystem start-up by, for example, centrifuging a portion of the selecteddonor's blood, or by initially using another biocompatible, non-toxicliquid until a depleted flow one (substantially or predominantly plasma)is generated at outlet 350.

Not separately illustrated in FIG. 3, an additional holographic trappingseparation stage may also be utilized to aid in the separation of theseblood fractions. For example, a holographic trapping separation stagemay be utilized in lieu of or in addition to, second stage 310. Inaddition, while the second stage separation has been illustrated usingflow one, in other embodiments, flow two may be subjected to a second(or more) stage separation, in addition to or in lieu of the additionalseparation stage of flow one. Moreover, additional separation stages maybe utilized in series or in parallel.

More generally, separation regions are regions where materials arepartially or fully sorted or separated based upon some materialproperty. The separation regions may employ one or more of the followingtechniques, individually, serially, or simultaneously, in any of thevarious flows in the embodiments of the invention:

-   -   (a) Sedimentation Rate Zonal Separation: separation by        sedimentation rate. The sedimentation rate is generally a        function of the material's size and density, as well as the        material's shape and electrostatic properties. For this        separation, laminar flow is set up and separation occurs under        gravity, or in some cases through other inertial forces such as        spinning in a centrifuge-like device.    -   (b) Isopycnic Separation: separation by density. For this        separation, a linear, step, smoothed-step, or alternate density        gradient is established and the materials are allowed to        sediment and/or cream in the gradient until the material reaches        or nearly reaches an area where the material is in an        environment of matched density.    -   (c) Diffusivity Separation: separation by diffusivity. For this        separation, materials are fractionated based upon the distance        they diffuse in a given amount of time.    -   (d) Motility Separation: separation by motility. In this        separation, materials are fractionated based on the distance the        material travels under its own motility in a given amount of        time.    -   (e) Optical Fractionation: separation using optical forces,        typically without feedback mechanisms to inform and influence        the optical system based upon investigation of the individual        objects.    -   (f) Direct Optical Separation: separation using optical forces,        typically using feedback mechanisms to inform and influence the        optical system based upon investigation of the individual        objects.    -   (g) Dielectrophoretic Separation: separation using        dielectrophoresis. The forces exerted upon an object depends        upon its position in the imposed electric field and the        dielectric response of the material and its environment.    -   (h) Electrophoretic Separation: separation using        electrophoresis. The fortes exerted upon an object depends upon        its position in the imposed electric field and the charge of the        material and its environment.    -   (i) Magnetic Separation: separation using magnetic forces. The        forces exerted upon an object depends upon its position in the        imposed magnetic field and the magnetic properties of the        object.    -   (j) Surface Tension Separation: separation using the surface        tension or surface chemistry of a material. This may, for        example, involve the creating of fluid interfaces which certain        materials may be attracted to or stable at.

More particularly, exemplary separations for blood sorting include: (a)separation of one or more blood cell types from some or all of the otherblood cell types and/or from the blood plasma or fluid medium bysedimentation rate zonal separation; (b) separation as in (a) but withisopycnic separation; (c) removal of just the red blood cells (RBCs) orthe RBCs and white blood cells (WBCs) from the solution bysedimentation; (d) concentration of the platelets from the plasma usingsedimentation; (e) extraction of the RBCs using dielectrophoresis,electrophoresis, or magnetic separation; (f) concentration or extractionof platelets from a solution using optical techniques including opticaltweezers and optical fractionation; and (g) separation of bloodcomponents using agents which may bind to a particular cell type (suchas functionalized beads) and be acted upon by any of the aboveseparation techniques, after which the agent may or may not be unboundfrom the cell type.

For blood sorting, a combined approach may be the most effective, suchas: first extract most of the RBCs and WBCs using sedimentation ratezonal separation, then extraction and concentration of the plateletsusing optical fractionation, discussed below. The optical fractionationstep will act not only to concentrate the platelets (which could also,for example, be done by a centrifugation step at the end), but willprovide a second step which will exert strong suppression on the WBCsaccidentally collected with the platelets, for the example of plateletaphoresis. Such concentration steps may also include filtering, such asto filter WBC from a platelet fraction or a plasma fraction.

While apparatus 300 has been described with respect to bloodfractionation, it will be understood by those of skill in the art thatthe apparatus 300 may be utilized for a wide variety of separations, inaddition to such blood fractionation. In addition, apparatus 300 mayalso be considered one particular embodiment of series-connectedseparation stages of the present invention.

Cell “washing” is also a significant application of the apparatus 100,200 or 300. Such washing may include a change of media, for storage,preservation, or other medical purposes. Such washing may also consistof removing a media containing contaminants such as bacteria, byseparating the cells of interest into another media flow free of suchcontamination. As indicated above, sperm separation is also asignificant application of the apparatus 100, 200 or 300.

Portions of, or outputs from, the sorting device 100, 200 or 300 may beinspected optically. This may be direct visual imaging, such as with acamera, utilizing direct bright-light imaging or fluorescent imaging.Or, it may be more sophisticated techniques such as spectroscopy,transmission spectroscopy, spectral imaging, or scattering such asdynamic light scattering or diffusive wave spectroscopy. In many cases,these inspection regions may be incorporated directly into the flowdevice to characterize the inputs, outputs, or intermediate steps. Theymay be for diagnostics or record-keeping, or they may be used to informthe overall process, such as for feedback on how processing is done oron the speed of flow or amount of each solution to use. In some cases,the optical inspection regions may be used in conjunction withadditives, such as chemicals which bind to or affect parts of thesolution or beads which are functionalized to bind and/or fluoresce inthe presence of certain materials or diseases. For the example of bloodsorting, these techniques may be used to measure cell concentrations, todetect disease, or to detect other parameters which characterize theblood.

Portions of, or outputs from, the sorting device 100, 200 or 300 alsomay be characterized electronically. For example, a portion of thesorting device may have electronic devices embedded. Example electronicdevices may include: (a) capacitors, (b) electronic flow meters, (c)resistance meters for determining the bulk conductivity of the fluid,from which concentrations or compositions may be measured, or (d) pHmeasuring devices. For the application of blood sorting, measurements ofcell concentration, iron content, flow rates, total cell counts,electrolyte concentration, pH, and other parameters may be a valuablepart of a sorting device.

The flow components of the sorting device 100, 200 or 300 may bepassive, being completely controlled externally by the flow rates of theinputs and outputs. Alternatively, there may be active flow componentsembedded in the device (not separately illustrated), such as valveswhich may be partially or fully opened or closed using electronic,optical, thermal, mechanical, or other influence. For the application ofblood sorting, the sorting device 100, 200 or 300 may have an integratedmethod for storing and/or delivering one or more solutions. For example,a consumable sorting device may be manufactured to have a deformablemembrane on a side of a reservoir. This reservoir may be filled with asolution, such as a buffering agent which is biologically compatiblewith the patient and which may be used to dilute the blood. Anotherexample is that it could be filled with an anti-coagulant. The deliveryand/or use of one such fluid may be actively controlled by mechanicalinfluence, pressing on the membrane to deliver the fluid. Alternately,another mechanism may be used to deliver the fluid. Of particularinterest, for the sake of simplicity and cost-saving, is the integrationof various fluid solutions needed at differing steps in the sorting.Integrating these components may result in substantial simplificationand reduction of the total cost of ownership and operation. It may alsoreduce the risk of contamination and error. Such reservoirs holdinginput and buffering solutions or fluids are illustrate, for example, inFIGS. 14 and 15.

The device 100, 200 or 300 may include areas (not separatelyillustrated) where biological or chemical investigation of one or moreof the fluids or fluid components. This may include measurements of pH,the presence of certain biological or chemical materials, or othermeasurements. For the application of blood sorting, this may includedetection of disease, characterization of concentrations of various celltypes or materials in the plasma, characterization of iron content,determination of blood type, or other evaluation of blood quality, type,or category.

The device 100, 200 or 300 may include a region (not separatelyillustrated) which sterilizes the solution using optical methods, suchas exposure to UV light, or other methods. The sterilization may actupon solutions which are initially part of the device or added to it forbuffering, washing, diluting, or other impacts on the sample solution.Or, the sterilization may act upon part or all of the sample solutionbeing processed. For the application of blood sorting, opticalsterilization of the solutions used in the device other than whole bloodmay be important. Also, sterilization of the blood or certain fractionsof the blood may be important.

The device 100, 200 or 300 may be comprised of materials such that oneor more surfaces have been constructed so as to interact physically orchemically with certain materials. For example, a surface may befunctionalized so that certain materials adhere to it, for the purposeof extracting these materials from the solution or for the purpose ofdiagnostics. For the application of blood sorting, functionalizedsurfaces may be used to extract unwanted materials from certainfractions. Alternately, they may be used to collect materials which areat low concentration for the purpose of measuring the degree to which amaterial or a type of material is present, such as for diseasedetection.

The sorting device 100, 200 or 300 may contain regions where sortingacts in parallel but without physical walls to separate the flows. Forexample, a parallel sorting region may have multiple inlets 120 andoutlets 130, some of which are functionally similar to each other.Instead of having physical dividers distinguishing the multiple parallelsorters, the division occurs as a consequence of the physical propertiesof the solutions and the flow. The contacting parallelized sortersregions may yield high sorting rates with more simple and cheap devices.They may also act to avoid contact with surfaces. For blood sorting,these regions would be used to minimize contact with surfaces and tomaximize sorting rate while reducing costs and complexity.

The device 100, 200 or 300 may have regions which are designed toregulate flow rate or the flow character (not separately illustrated).For example, in many cases laminar flow is required, and often aparticular flow profile is desired. In other cases, several regions ofthe device should have identical flow rates and behavior. For thesereasons, areas with shapes and other properties to influence flowbehavior are often needed. In some cases, this is done by making verysymmetric flow designs. In other cases, large changes in the diameter offlow regions and/or the existence of reservoirs help to maintain uniformflow rates. In other cases, very carefully designed channels provide theexact balancing of flow rates needed. To maintain laminar flow, areaswhere slow changes in channel size occur may be important. Obstacles ordividers may also act to maintain laminar flow. For blood sorting, flowregulation is important to achieve high sorting rates while maintainingthe yield and purity of the fractions.

Not separately illustrated, the device 100, 200 or 300 may containregions which are designed for mechanical mixing of the fluids, such asregions which encourage turbulence. For example, a region with a fastnarrow stream entering a region with a large dimension may produceturbulent flow and mixing. For blood sorting, a mixing region may mix adiluent or anticoagulant with the blood, or may mix other solutionstogether as needed.

The device 100, 200 or 300 may contain regions which align cells ormaterials in a certain way (not separately illustrated). This issometimes done through shear flows, but may also be done by imposingexternal fields such as electric fields.

The device 100, 200 or 300 may contain regions designed to lyse cells orbreak up materials (not separately illustrated). This may be donethrough shear flows, vibration, forcing through an orifice, electrical,or other means. For blood sorting, this may be valuable for theelimination of certain cell types or aggregates which may form. It mayalso be valuable for diagnostic purposes, such as disease detection ormeasurement of parameters which pertain to the contents of cells.

The device may contain regions (not separately illustrated) which swellor dehydrate cells or objects, such as by introducing agents whichchange the osmotic pressure or by changing the physical pressure. Thismay be done, for example, as a step prior to isopycnic sorting to adjustthe density of the cells or objects. It may also be done to kill orshock certain components. For the example of blood sorting, this may bedone as a later stage purification step to remove or neutralizeundesired components.

The device 100, 200 or 300 may contain regions which heat or cool one ormore solutions (not separately illustrated). This may be done for itsimpact on physical properties, such as viscosity or density. Or, it maybe done for its impact on chemical properties, such as chemical reactionrates or chemical stability. Or, it may be done for its impact onbiological properties, such as motility, metabolism rate, or viability.For example, one fluid flow may be at a higher temperature than another,causing motile sperm to move away from the hotter fluid to the coolerfluid. Also, it may be done for system-level compatibility, such as inpreparation for the following processing step. For the example of bloodsorting; solutions which are returned to the patient may be maintainedat an appropriate temperature to avoid chilling the patient. Solutionswhich are to be stored may be cooled during processing to preserve thosefractions or prepare them for the next processing or storage step.

The device 100, 200 or 300 may contain reservoirs which serve to storesolutions which will be used during the process run, or which may begenerated during the process run (not separately illustrated). Havingthese reservoirs integral to the sorting device simplifies the use ofthe device and reduces the need for additional parts. For the example ofblood sorting, reservoirs may contain anticoagulants, diluents,dilutants, and any other solutions needed in the process. Reservoirs mayalso be incorporated which will hold the sorted fractions or wastefractions.

The device 100, 200 or 300 may contain regions which enhance mixing bydiffusion (not separately illustrated). For example, when mixing bycontacting two laminar flows, parallelizing into many narrow contactingflows enhances the overall mixing rate by diffusion. For the example ofblood sorting, diffusive mixing regions may be used to mix diluent,anticoagulant, or other solutions with whole blood or blood fractions.

Also not separately illustrated, the device may contain regions withbubble traps to remove air bubbles from the system. This may be done byhaving a region where air bubbles are able to rise from a region withflow to a region above the primary flow region. For the example of bloodsorting, this may be done in a simple way to guarantee that small airbubbles from the loading or running of the system do not pass on to thepatient or the collection samples.

As indicated above, the device 100, 200 or 300 may contain regions whichact to suppress any pulsation in the flow (not separately illustrated),such as that which occurs when peristaltic pumps are used. One way tosuppress pulsation is to incorporate a “bubble-trap” into the device.The presence of an air pocket, which is in contact with the fluid,allows for compression of the air pocket as pressure in the fluidincreases and decreases. Thus, the air pocket acts as a shock absorber,smoothing out the flow. Other devices may be used as well, such as aflexible membrane which may bend under higher pressures, therebysmoothing out the pressure and flow rate. For the application of bloodsorting, pulsation reduction regions will yield more precise and smoothflows, and therefore higher sorting rates, purity, and yield.

The device 100, 200 or 300 may contain regions which reveal the state ofthe device (not separately illustrated). For a consumable, it mayindicate whether the device has been sterilized or whether it has beenused. For the application of blood sorting, one would want indicators toconfirm both that the sorting device has been sterilized and that thedevice has not yet been used or contaminated.

The sorting device 100, 200 or 300, or overall sorting system, maycontain mechanisms for precise leveling of the flow sorter. This isimportant because for some sorters, buoyant forces may causeunintentional flows and have negative impacts on sorting yields andpurity in cases where the device is not precisely leveled. Additionally,the sorting device may contain components which reveal whether it iswell-balanced, or the degree to which it is balanced. For example, itmay have an electronic or gravity-based balance incorporated in thesorting device itself. One example of such a device is a shaped channelwith fluid and an air bubble in it. The position of the air bubble mayreveal the angle of the tilt of the device. Another such device may usea metal ball in a track to reveal the tilt angle. Another manifestationis to use an optical alignment, such as bouncing a light source off asurface or passing a light source through a wedge to identify itsorientation. For the application of blood sorting, leveling controls andindicators are significant to guarantee high-yield and high-purityproducts.

The sorting device 100, 200 or 300, or overall sorting system, maycontain mechanisms for maintaining uniform and/or constant temperaturesof the device and/or the solution (not separately illustrated). This isimportant to eliminate thermally-induced buoyant forces which may causeunintentional flows and have negative impacts on sorting yields andpurity. Additionally, the sorting device may have indicators in it, orin the sorting system as a whole, which indicate the temperature and/ortemperature uniformity of one or more components. For the application ofblood sorting, temperature uniformity controls and indicators may besignificant to guarantee high-yield and high-purity products.

The sorting device 100, 200 or 300, or overall sorting system, maycontain mechanisms for measuring the level and concentration of one ormore input or output solution (not separately illustrated). Thesemeasurements may be used to gauge the speed of operation, completiontime, error state, for general monitoring, or for other applications.For the application of blood sorting, level and concentration indicatorsmay be used to identify when sufficient sample has been collected or todetect when a failure or depletion of a solution has occurred.

Lastly, the sorting device 100, 200 or 300, or overall sorting system,may have a method for priming the system with one or more fluids usingstandard bottom-up filling or evacuation. Purging may similarly be doneby draining the device or by flowing a solution through it. At the earlystages of a sorting run, the priming solution may be discarded until atime when the priming solution has been mostly exhausted and the desiredsolution is obtained. Similarly, at the late stages of a sorting run, afluid may be used to push the sorted material through the system tominimize waste and maximize yield.

The various sorting devices 100, 200 or 300 may also be utilized in asystem providing a general purpose device which allows a user to extractone or more fractions of a solution, determined by a range of S values(size, density, or size*density). FIG. 26 is a block diagramillustrating an exemplary sorting and separation system 2600 inaccordance with one embodiment consistent with the present invention.This general purpose sorter would vary the flow rates on each input andoutput channel in response to user controls (user input 2610). The usercontrols would allow the user to determine the range of S values thatleave through each output channel. This device would be a valuable labtool for performing various purifications, washing, separations, anddiagnostic evaluations of samples. It would also be an importantplatform for research, development, and prototyping applications.

The sorting hardware may be contained within a single enclosure whichmay or may not be temperature controlled. This hardware consists of (1)Consumable flow plate 2620 (e.g., sorting device 100, 200 or 300) withseveral inputs and outputs, (2) several computer-controllableperistaltic pumps 2625, (3) temperature control and monitoring apparatus2630, (4) reservoir holders (or reservoirs 2635 and 2640). The sortingis controlled by a computer 2650 which monitors and controls the flowrates, temperature, and any other additional components which may beincluded for controlling or diagnostics purposes.

The flow plate 2620 may be a simple general-purpose device, suitable formany applications, as discussed above. It may have two to four inputsand two to ten outputs. It may be provided in a sterile, primed, andsealed state. The reservoirs may be part of the flow plate, or they maybe separate sterile components which are attached to the flow platebefore use. All or a portion of the central sorting region may becovered with a cover glass to allow manipulation with optical tweezers,optical dielectrophoresis, or laser killing/cutting of samples.

The computer 2650 control does measurements and control of the hardwarefor pumping and temperature control. It may also interface otherhardware components that may be added. The user software provides a veryconvenient front end to the simultaneous control of all the pumps. Itwould do the necessary math to control each pump rate to give the userthe desired flows for the particular sorting application.

This system 2600 would be general enough to allow for many sortingapplications, either independently or in conjunction with an additionallaser apparatus. These applications include but is not limited to: cellidentification by fluorescence, cell killing by high intensity laserexposure, cell fractionation using optical dielectrophoresis, motilecell fractionation based on motility, passive sorting of objects bydiffusivity, and passive fluidic zonal density sorting. The passivefluidic zonal density sorting applications, which are numerous andextend over a broad range, can generally be done with minimal or noadditional hardware.

Exemplary uses and applications for such a general purpose sortingsystem 2600, using passive fluidic sorting and/or use of opticalgradient forces (discussed below) are outlined in the following table:

Category General Specific Washing - Removing Cell Washing for DiseaseRemoving debris, bacteria, and viruses from Components Removal humansperm Removing debris, bacteria, and viruses from animal sperm AggregateRemoval Removing undesirable clumps of materials in industrial processesRemoving aggregated colloids in solution Removing coarsened droplets inan emulsion Precursor Removal Removing precursor materials from a haltedgrowth process Separating differentiated cells Removing blastocytes fromincubating sperms and eggs Washing - Changing Cell Processing Changingosmotic conditions - Cell swelling or Media shrinking Cell stainingAutomated multi-step cell experiments or processing Sperm fluoridationto suppress activity Material Processing Bead dying AssaysObject-solution response assays Diagnostics Characterizing MonodisperseMeasuring S (Svedberg coefficient) of particles Solutions Measuring sizeof particles of known material Measuring density of particles of knownsize Measuring composition of particles Characterizing PolydisperseMeasuring distribution of S, size, density, Solutions composition, etc.Measuring average S, size, density, composition, etc. Disease DetectionExtracting a sample of bacteria from infected tissue Extracting a sampleof bacteria from stool samples Extracting a sample of bacteria fromblood Extracting cells that are infected with viruses from healthy cellsEnvironmental Extracting spores from sample to determine spore count inair Extracting particulate matter from water or air for environmentalmonitoring Monitoring water safety in drinking or swimming waterExtracting spres from environmental samples to quantify mold levels ininfected homes Food Safety Extracting a sample of bacteria from food fordiagnosis Purifying - Reducing Viruses Purifying a virus sample VariancePurifying - Extracting Cell Components Separating different cellcomponents from Components solution of lysed cells Cell ComponentsSeparating organelles Bacteria Extracting a sample of bacteria frominfected tissue Spores Extracting a sample of spores from a solutionFiltering Filtering Large Components Removing yeast from beer From FoodsRemoving yeast from wine Removing fat from milk Industrial FilteringRemoving particulates from water Removing particulates from machine oilPharmaceutical Filtering Removing undissolved clumps from solution toprevent overdosing Medical Filtering Dialysis, using a membrane, forkidney dialysis Sorting Cell Type Sorting Sorting blood cells from bloodto extract the plasma Sorting platelet cells from blood for aphoresisSorting cells based on presence of given antibody (may usefunctionalized beads) Sorting natural killer cells from blood as atreatment for AIDS Sorting different types of cells within a tissue ororgan (eg. sorting osteoclasts, osteoplasts, osteoblasts) Sorting whiteblood cells as a treatment for white blood cell diseases (e.g., high WBCsuch as leukemia or lymphomia, or low WBC) Sorting sickle cells fromnormal red blood cells as a treatment for sickle-cell anemia CellCluster Sorting Sorting islet cell clusters by size (diabetes) Removingcell clusters from single cells Cell State Sorting Removing infectedcells from healthy cells Removing living cells from dead cellsSeparating proliferating and non-proliferating cells Isolating viablesperm from inviable sperm Isolating Cells From Biopsies Purifying bonemarrow cells from blood in marrow biopsy Colloids Fractionation Sortingcolloids by size, density, composition, S, etc. Sorting Variants of OneCell Sorting multizygotic sperm from normal sperm Type Removing mostsevere RBCs in sickle cell anemia patients

FIG. 27 is a block diagram illustrating an exemplary bioreactor productpurification and separation system 2700 in accordance with oneembodiment consistent with the present invention. A bioreactor is usedin the production of monoclonal antibodies, recombinant proteinproducts, viruses and viral antigens, and viable cell mass. The mostcommon use for a bioreactor is in the production of various drugtherapies. A bioreactor basically works by allowing cells to grow inhigh concentrations in ideal conditions. While the cells are growing aculture medium is flowed through the bioreactor. This medium collectsthe cell's waste products, as well as provides nutrition for the cells.The medium that has been flowed through the bioreactor is then processedfor the product. In order to collect the product, the waste filledmedium is filtered through many different processes, one of which isthrough bead column chromatography. This process takes a long time tocomplete, is very expensive, and does not yield a large percentage ofthe final product. The present invention solves all three of theseproblems.

The bioreactor product purifier 2700 would quickly, easily, andaccurately remove the wanted product from the waste medium. The wastemedia would be flowed into the sorter through one channel 2701 At thesame time a solution of beads coated with the proper affinity siteswould be flowed into the sorter through the second channel 2702. Bothchannels would lead to a first mixer 2705 in which they would be mixedtogether. During the mixing the product would bind to the sites on thebeads. The mixture would then flow through a first separation region2710 along with an input buffer (input channel 2715). Through the use ofpassive diffusive sorting, the beads with selected product outputthrough channel 2718 and the waste solution would be output throughchannel 2716 and discarded. Passive diffusive sorting succeeds becausethe large beads stay in one channel while the lighter molecules diffuseto the other side of the channel. The bead solution is then be flowedthrough channel 2718 as a denaturing solution is flowed through a secondchannel 2720. Both of these channels would flow to a second mixer 2725where they would be mixed together. While they were being mixed, thedenaturing solution would break the bond between the product and thebeads. After mixing, the solution would be flowed through an outputchannel 2735 along with another buffer solution through channel 2730,into second separation region 2740. Through the use of passive diffusivesorting, the beads would be flowed through the bottom channel 2745 andbe discarded as waste. The purified and recovered product would beflowed through the top channel 2750. This method of purification shouldreduce costs, reduce time, and increase the yield of final product.

FIG. 4 schematically illustrates a holographic optical trapping system400, generally used in conjunction with an apparatus 100, 200 or 300,according to one embodiment consistent with the present invention.Additional detail concerning holographic optical trapping is availablein the fifth related application. In a holographic optical trappingapparatus or system 400 as illustrated in FIG. 4, light is incident froma laser system, and enters as shown by the downward arrow, to power thesystem 400.

A phase patterning optical element 401 is preferably a dynamic opticalelement (DOE), with a dynamic surface, which is also a phase-onlyspatial light modulator (SLM) such as the “PAL-SLM series X7665,”manufactured by Hamamatsu of Japan, the “SLM 512SA7” or the “SLM512SA15” both manufactured by Boulder Nonlinear Systems of Lafayette,Colo. These dynamic phase patterned optical elements 401 arecomputer-controlled to generate beamlets by a hologram encoded in themedium which may be varied to generate the beamlets and select the formof the beamlets. A phase pattern 402 generated on the lower left of FIG.4 produces the traps 403 shown in the lower right filled with 1 μmdiameter silica spheres 404 suspended in water 405. Thus, the system 400is controlled by the dynamic hologram shown below on the left.

The laser beam travels through lenses 406, 407, to dichroic mirror 408.The beam splitter 408 is constructed of a dichroic mirror, a photonicband gap mirror, omni directional mirror, or other similar device. Thebeam splitter 408 selectively reflects the wavelength of light used toform the optical traps 403 and transmits other wavelengths. The portionof light reflected from the area of the beam splitter 408 is then passedthrough an area of an encoded phase patterning optical element disposedsubstantially in a plane conjugate to a planar back aperture of afocusing (objective) lens 409.

In single beam optical trapping (also called laser or optical tweezers)it had been thought, prior to the invention of the fifth relatedapplication, that a high numerical aperture lens was necessary foracceptable optical traps. A basis for this thinking was that, foroptical trapping, one uses the gradient in the electric field of theimpinging light to trap the particle. In order to have a large trappingforce it has been thought necessary to have a large gradient in theelectric field (or number density of rays). The way that one usuallyaccomplishes this is to pass the light field through a high numericalaperture lens.

A concern with observation and trapping of samples within a large fieldof view is that such observation and trapping would involve an objectivelens with a low numerical aperture. Contrary to prior teaching, theinvention of the fifth related application provides a low numericalaperture lens as, for example, the objective lens 409 in FIG. 4. Theability to observe and trap in this situation could be useful in anyapplication where one would benefit from a large field of view given bya low magnification lens, such as placing microscopic manufactured partsor working with large numbers of objects, such as cells, for example.

As an example according to the present invention, 3 micron silicaspheres 104 suspended in water 105 were trapped with lenses 109 with anunprecedented low numerical aperture. The lenses 109 used weremanufactured by Nikon: (a) Plan 4× with an NA of 0.10; and (b) Plan 10×with an NA of 0.25.

Suitable phase patterning optical elements are characterized astransmissive or reflective depending on how they direct the focused beamof light or other source of energy. Transmissive diffractive opticalelements transmit the beam of light or other source of energy, whilereflective diffractive optical elements reflect the beam.

The phase patterning optical element 401 may also be categorized ashaving a static or a dynamic surface. Examples of suitable static phasepatterning optical elements include those with one or more fixed surfaceregions, such as gratings, including diffraction gratings, reflectivegratings, and transmissive gratings, holograms, including polychromaticholograms, stencils, light shaping holographic filters, polychromaticholograms, lenses, mirrors, prisms, waveplates and the like. The static,transmissive phase patterning optical element is characterized by afixed surface.

In some embodiments, however, the phase patterning optical element 401itself is movable, thereby allowing for the selection of one more of thefixed surface regions by moving the phase patterning optical element 401relative to the laser beam to select the appropriate region.

The static phase patterning optical element may be attached to a spindleand rotated with a controlled electric motor (not shown). The staticphase patterning optical element has a fixed surface and discreteregions. In other embodiments of static phase patterning opticalelements, either transmissive or reflective, the fixed surface has anon-homogeneous surface containing substantially continuously varyingregions, or a combination of discrete regions, and substantiallycontinuously varying regions.

Examples of suitable dynamic phase patterning optical elements having atime dependent aspect to their function include computer-generateddiffractive patterns, phase-shifting materials, liquid crystalphase-shifting arrays, micro-minor arrays, including piston modemicro-mirror arrays, spatial light modulators, electro-optic deflectors,accousto-optic modulators, deformable mirrors, reflective MEMS arraysand the like. With a dynamic phase patterning optical element 401, themedium 405 which comprises the phase patterning optical element 401encodes a hologram which may be altered, to impart a patterned phaseshift to the focused beam of light which results in a correspondingchange in the phase profile of the focused beam of light, such asdiffraction, or convergence. Additionally, the medium 405 may be alteredto produce a change in the location of the optical traps 403. It is anadvantage of dynamic phase patterning optical elements 401, that themedium 405 may be altered to independently move each optical trap 403.

In those embodiments in which the phase profile of the beamlets is lessintense at the periphery and more intense at regions inward from theperiphery, overfilling the back aperture by less than about 15 percentis useful to form optical traps with greater intensity at the periphery,than optical traps formed without overfilling the back aperture.

In some embodiments, the form of an optical trap may be changed from itsoriginal form to that of a point optical trap, an optical vortex, Besselbeam, an optical bottle, an optical rotator or a light cage The opticaltrap may be moved in two or three dimensions. The phase patterningoptical element is also useful to impart a particular topological modeto the laser light, for example, by converting a Gaussian into aGauss-Laguerre mode. Accordingly, one beamlet may be formed into aGauss-Laguerre mode while another beamlet may be formed in a Gaussianmode. The utilization of Gauss-Laguerre modes greatly enhances trappingby reducing radiation pressure.

1. Imaging System

The current instrument design uses a high resolution CCD camera for theprimary imaging system 110. The main advantage of the CCD camera (seereference numeral 511 in FIG. 5) is the favorable cost/performance ratiosince this technology is a mature one. Another advantage of CCD camerasis their wide dynamic range and the ease of generating digital output.

The images are viewed on a computer screen (see reference numeral 510 inFIG. 5) to provide both a frame of reference for selecting the locationof the traps as well as to minimize the possibility of inadvertentexposure of the operator to the laser.

2. User Interface

a. Object Display

The user interface consists of a computer screen which displays thefield of view acquired by the CCD camera. The user designates the lociof the traps with a mouse. There is also an option to delete a location.

As described in greater detail below, the user is also able to specifythe power per trap so as to be able to avoid specimen damage. Inaddition it is desirable to be able to vary trap power because trappingdepends upon the difference between the index of refraction of thespecimen and the suspending medium which can be expected to vary fromspecimen to specimen.

b. The Hologram

The purpose of designating the loci of the traps is to provide input forthe hologram calculation. The hologram is essentially a function whoseFourier transform produces the desired trap array. However in the caseof the liquid crystal display this function is a phase object (i.e., anobject that changes the phase of the wavefront without absorbing anyenergy).

c. Methods for Choosing the Set of Traps

Often one wishes to use the traps to move an object in a particulardirection. This may be accomplished by using the mouse to create a line(by dragging). The computer program interprets a line as calling for aseries of traps to be deployed sequentially and sufficiently closetogether so as to move the target in small steps without losing the lockon the target.

The present invention also includes the capability of changing theheight of the traps. If a laser beam is parallel to the optical axis ofthe objective lens 409, then a trap forms at the same height as thefocal plane of the lens 409. Changing the height of a trap isaccomplished by adjusting the hologram so that the beam of light forminga trap is slightly converging (or diverging) as it enters the objectivelens 409 of the microscope. Adjusting the height of a trap is possibleusing lenses but only a holographic optical trapping (HOT) allows theheight of each individual trap to be adjusted independently of any othertrap. This is accomplished by the computer program adjusting the phasemodulation caused by the liquid crystal hologram.

3. Sample Holder

a. General

The sample chamber 700 (see FIGS. 7A and 7B) of the present invention isinexpensive and disposable. Although the sample chamber 700 of thepresent invention is described below, another object of the presentinvention is to create a flexible design that may be changed fordiffering applications. In addition to the sample chamber 700, thevarious other separation stages of the invention may be utilized, suchas an apparatus 100, 200 or 300, or the other separation stagesdiscussed below with reference to FIGS. 13-24 and 26-27.

The sample chamber 700 lies on the surface of a microscope slide 701.The sample chamber 700 contains a series of channels 703 for introducingspecimens or objects. The channels 703 are connected to supply andcollection reservoirs by thin tubing 704 (commercially available).Samples or objects will be suspended in a liquid medium and will beintroduced into the working area via the channels 703. The samplechamber 700 is covered by a cover slip 705.

b. Manufacture of the Sample Chamber

In one embodiment consistent with the present invention, a poly(dimethylsiloxane) (PDMS) resin is used to fabricate the chamber 700. The processinvolves creating the desired pattern of channels 703 on a computerusing standard CAD/CAM methods and transferring the pattern to aphotomask using conventional photoresist/etching techniques. Thephotomask is then used as a negative mask to create an inverse patternof channels which are etched on a silicon wafer. The depth of thechannels 703 is controlled by the etch time. The silicon wafer is anegative replica of the actual sample chamber 700. The final stepconsists of creating the positive sample chamber 700 by pouring PDMSonto the wafer and polymerizing. This results in a PDMS mold which isbonded to a glass slide 701 and overlaid with a cover slip 705. Theglass to PDMA bonding is effected with an oxygen etch which activatesthe exposed surfaces.

A number of additional steps are necessary to ensure consistent quality.For instance the PDMS solution/hardner is maintained under a vacuum inorder to prevent bubble formation. The silicon wafer is silanized toprevent the PDMS from sticking to the wafer. There are a variety ofsteps involving cleaning the replicas and maintaining properenvironmental controls. These represent standard technology.

The channels 703 are connected to microbore tubing 704 using smallsyringe needles 706 held using glue 714, which are inserted through thePDMS mold into small circular wells 707 which connect to each channel703. Sample solutions are introduced into the channel 703 usingmicropumps 708.

FIG. 7B shows a diagram of a typical arrangement for the introduction ofa sample via the syringe pump 708 at 710. The medium is introduced at711, and waste is collected at 71 and the desired collections at 713.

FIG. 8 presents a representation of a scanning electron micrograph ofthe diagram in FIG. 7B as actually created from the process describedabove. The channels are approximately 50 microns wide and 50 micronsdeep. FIG. 9 presents a representation of a scanning electron micrographof the ‘working’ volume where manipulations of the specimen under studywould occur. The diagrams clearly show that the channels 703 are smoothand clean. Although the channels 703 are rectangular in cross-section,other shapes may be devised as well. The channels 703 are designed toallow samples to be flowed to a ‘working area’ whose shape may be customdesigned for experimental requirements.

c. Holographic Optical Traps

Unlike scanned optical traps which address multiple trapping points insequence, and thus are time-shared, holographic optical traps illuminateeach of their traps continuously. For a scanned optical trap to achievethe same trapping force as a continuously illuminated trap, it mustprovide at least the same time-averaged intensity. This means that thescanned trap has to have a higher peak intensity by a factorproportional to at least the number of trapping regions. This higherpeak intensity increases the opportunities for optically-induced damagein the trapped material. This damage may arise from at least threemechanisms: (1) single-photon absorption leading to local heating, (2)single-photon absorption leading to photochemical transformations, and(3) multiple-photon absorption leading to photochemical transformations.Events (1) and (2) may be mitigated by choosing a wavelength of lightwhich is weakly absorbed by the trapping material and by the surroundingfluid medium. Event (3) is a more general problem and is mitigated inpart by working with longer-wavelength light. Thus holographic opticaltraps may manipulate delicate materials more gently with greater effectby distributing smaller amounts of force continuously among a number ofpoints on an object rather than potentially damaging the object byexerting the total force on a single point or at a higher intensity fora period of time.

In one embodiment consistent with the present invention, the design isflexible in that any desired pattern of channels 703 may be designedwith a standard CAD/CAM computer program. The complexity of the patternis not a factor as long as the channels 703 are far enough apart so asnot to impinge on one another. As may be seen in FIGS. 7B and 8,multiple sets of channels 703 may be easily accommodated so that asingle chip may be used for more than one experiment. In addition, oncea mold is made it may be used to fabricate thousands of sample chambersso the methodology is readily adaptable to mass production techniques.It is estimated that the marginal cost of a single chamber would be ofthe order of a few cents when in mass production.

4. Optical System

a. Synthesizing the Hologram

Early versions of the holographic optical traps used fixed hologramsfabricated from a variety of materials. These were adequate todemonstrate the principle of using holograms to create up to severalhundred traps. However the major shortcoming of these holograms was thatthey were static and it took hours to make a single hologram. With theadvent of the hardware to create computer-driven liquid crystal displayscapable of forming holograms many times per second, the use of opticalimps as a dynamic device has become a practical reality. Softwarecontrol permits automation of separation and trapping by simpleimplementation of programs to control laser beam steering. The principlefor computing the hologram is described below.

b. The Microscope

The optical system 410 consists of a standard high quality lightmicroscope. The objective is a high numerical aperture lens 409 coupledwith a long working distance condenser lens. The high numerical apertureobjective lens 409 is used for trapping. While the long working distancecondenser lens may somewhat reduce the resolution in the images, it doesnot compromise trapping and provides extra space near the sample slideto accommodate plumbing and receptacles. The objects may be moved byholding them with traps and moving the stage of the microscopevertically or laterally.

In one embodiment consistent with the present invention, approximately 2mW of laser power is employed to produce 200 microwatts at the trap. Thepower level available from a 2 W laser is adequate to create about 1000traps. A green laser (532 nm) is used, but other wavelengths may also beused, including, for example, a far red laser to work with materialsabsorbing near the 532 nm value.

Trapping depends upon the refractive index gradient so that materialswith refractive indices close to that of the surrounding medium needtraps with higher power levels. In addition, the tolerance of materialsto damage will vary with trap power, so it is desirable for the user tobe able to control this parameter. The user may increase the power levelin any particular trap using a ‘power slider’ displayed on the graphicalinterface.

c. The Liquid Crystal Hologram (Also Referred to as a Spatial LightModulator or SLM)

The spatial light modulator 408 is essentially a liquid crystal arraycontrolled by an electrostatic field which, in turn may be controlled bya computer program. The liquid crystal array has the property that itretards the phase of light by differing amounts depending upon thestrength of the applied electric field.

Nematic liquid crystal devices are used for displays or for applicationswhere a large phase-only modulation depth is needed (2Π or greater). Thenematic liquid crystal molecules usually lie parallel to the surface ofthe device giving the maximum retardance due to the birefringence of theliquid crystal. When an electric field is applied, the molecules tiltparallel to the electric field. As the voltage is increased the index ofrefraction along the extraordinary axis, and hence the birefringence, iseffectively decreased causing a reduction in the retardance of thedevice.

d. The Laser

Useful lasers include solid state lasers, diode pumped lasers, gaslasers, dye lasers, alexandrite lasers, free electron lasers, VCSELlasers, diode lasers, Ti-Sapphire lasers, doped YAG lasers, doped YLFlasers, diode pumped YAG lasers, and flash lamp-pumped YAG lasers.Diode-pumped Nd:YAG lasers operating between 10 mW and 5 W arepreferred. The preferred wavelengths of the laser beam used to formarrays for investigating biological material include the infrared, nearinfrared, visible red, green, and visible blue wavelengths, withwavelengths from about 400 nm to about 1060 nm being most preferred.

FIG. 5 is a schematic diagram of a holographic optical trapping systemfor sorting objects, and is used in conjunction with an apparatus 100,200 or 300, according to one embodiment in accordance with the presentinvention. In one such embodiment, an optical trapping system 500 (seeFIG. 5) (such as the BioRyx system sold by Arryx, Inc., Chicago, Ill.)includes a Nixon TE 2000 series microscope 501 into which a mount forforming the optical traps using a holographic optical trapping unit 505has been placed. The nosepiece 502 to which is attached a housing, fitsdirectly into the microscope 501 via the mount. For imaging, anillumination source 503 is provided above the objective lens 504 toilluminate the sample 506. In accordance with the present invention, thesample 506 is one of the separation stages of the apparatus 100, 200 or300.

In one embodiment, the optical trap system 400 (see FIGS. 4 and 5)includes one end of the first light channel which is in close proximityto the optical element, and the other end of the first light channelwhich intersects with and communicates with a second light channelformed perpendicular thereto. The second light channel is formed withina base of a microscope lens mounting turret or “nosepiece”. Thenosepiece is adapted to fit into a Nixon TE 200 series microscope. Thesecond light channel communicates with a third light channel which isalso perpendicular to the second light channel. The third light channeltraverses from the top surface of the nosepiece through the base of thenosepiece and is parallel to an objective lens focusing lens 409. Thefocusing lens 409 has a top and a bottom forming a back aperture.Interposed in the third light channel between the second light channeland the back aperture of the focusing lens is a dichroic mirror beamsplitter 408.

Other components within the optical trap system for forming the opticaltraps include a first mirror, which reflects the beamlets emanating fromthe phase patterning optical element 401 through the first lightchannel, a first set of transfer optics 406 disposed within the firstlight channel, aligned to receive the beamlets reflected by the firstmirror, a second set of transfer optics 407 disposed within the firstlight channel, aligned to receive the beamlets passing through the firstset of transfer lenses, and a second mirror 408, positioned at theintersection of the first light channel and the second light channel,aligned to reflect beamlets passing through the second set of transferoptics and through the third light channel.

To generate the optical traps, a laser beam is directed from a laser 507(see FIG. 5) through a collimator and through an optical fiber end 508and reflected off the dynamic surface of the diffractive optical element509. The beam of light exiting the collimator end of the optical fiberis diffracted by the dynamic surface of the diffractive optical elementinto a plurality of beamlets. The number, type and direction of eachbeamlet may be controlled and varied by altering the hologram encoded inthe dynamic surface medium. The beamlets then reflect off the firstmirror through the first set of transfer optics down the first lightchannel through the second set of transfer optics to the second mirror;and are directed at the dichroic mirror 509 up to the back aperture ofthe objective lens 504, are converged through the objective lens 504,thereby producing the optical gradient conditions necessary to form theoptical traps. That portion of the light which is split through thedichroic mirror 509, for imaging, passes through the lower portion ofthe third light channel forming an optical data stream (see FIG. 4).

Spectroscopy of a sample of biological material may be accomplished withan imaging illumination source 503 suitable for either spectroscopy orpolarized light back scattering, the former being useful for assessingchemical identity, and the later being suited for measuring dimensionsof internal structures such as the nucleus size. Using suchspectroscopic methods, in some embodiments, cells are interrogated. Acomputer 510 may be used to analyze the spectral data and to identifycells bearing either an X or Y chromosome, or a suspected cancerous,pre-cancerous and/or non-cancerous cell types, or identify various typesof blood cells, for example. The computer program then may apply theinformation to direct optical traps to contain selected cell types. Thecontained cells then may be identified based on the reaction or bindingof the contained cells with chemicals.

The present method and system lends itself to a semi-automated orautomated process for tracking the movement and contents of each opticaltrap. The movement may be monitored, via video camera 511, spectrum, oran optical data stream and which provides a computer program controllingthe selection of cells and generation of optical traps.

In other embodiments, the movement of cells is tracked based onpredetermined movement of each optical trap caused by encoding the phasepatterning optical element. Additionally, in some embodiments, acomputer program is used to maintain a record of each cell contained ineach optical trap.

The optical data stream may then be viewed, converted to a video signal,monitored, or analyzed by visual inspection of an operator,spectroscopically, and/or video monitoring. The optical data stream mayalso be processed by a photodetector to monitor intensity, or anysuitable device to convert the optical data stream to a digital datastream adapted for use by a computer.

In an approach which does not employ an SLM (spatial light modulator),movement is accomplished by transferring the objects from a first set ofoptical traps to a second, third, and then fourth etc. To move theobjects from the first position to a second position, a static phasepatterning optical element is rotated around a spindle to align thelaser beam with a second region which generates the second set ofoptical traps at a corresponding second set of predetermined positions.By constructing the second set of optical traps in the appropriateproximity to the first position, the probes may be passed from the firstset of optical traps to the second set of optical traps. The sequencemay continue passing the probes from the second set of predeterminedpositions to a third set of predetermined positions, from the third setof positions to a fourth set of predetermined positions, and from thefourth set of predetermined positions and so forth by the rotation ofthe phase patterning optical element to align the appropriate regioncorresponding to the desired position. The time interval between thetermination of one set of optical traps and the generation of the nextis of a duration to ensure that the probes are transferred to the nextset of optical traps before they drift away.

In a staggered movement of the objects from a wide to narrow proximitythe staggered movement of the cells occurs in a similar fashion.However, as the objects are passed from a first set of optical traps toa second set and moved to second and subsequent positions, the staggeredarrangement of the traps allows the objects to be packed densely withoutplacing a set of traps in too close a proximity to two objects at thesame time which could cause the objects to be contained by the wrongoptical trap

Once an object or cell has interacted with a trap, spectral methods maybe used to investigate the cell. The spectrum of those cells which hadpositive results (i.e., those cells which reacted with or bonded with alabel) may be obtained by using imaging illumination such as thatsuitable for either inelastic spectroscopy or polarized light backscattering. A computer may analyze the spectral data to identify thedesired targets and direct the phase patterning optical element tosegregate to those desired targets. Upon completion of the assay,selection may be made, via computer and/or operator, of which cells todiscard and which to collect.

Optical peristalsis (see FIG. 13) is an existing process employingparallel lines of traps 1300 in a microfluidic channel 1301 arranged sothat the spacing between the lines permits particles 1302 trapped in oneline to be pulled into traps in the other line when the first line oftraps is turned off. Optical peristalsis may be used as an alternativeto and in conjunction with fluorescent labels (as described laterregarding applications). The process operates by timing the extinctionof lines of traps timed so that particles are moved in desireddirections specified by the arrangement of the lines of traps. Bychoosing whether a line of traps on one side or the other of a particleare on or off, the particle may be moved forward or back in a direction.By employing large numbers of traps, large numbers of particles may thusbe moved in concert in a given direction. Thus, particles attracted tothe traps may be moved to a given area and, if desired, collected there.This process may also be utilized in the various fluid flows utilizedwith the apparatus 100, 200 or 300.

Similarly, by gradually reducing the spacing between traps in linestoward a given direction and/or varying the curvature of the lines oftraps, particles may be swept into a focusing pattern to concentratethem. Reversing such a pattern would disperse the particles.

Spacing between lines of traps may be relatively larger to speed upmovement of the particles, or relatively narrower to slow them down.Similarly, varying the intensity of selected traps or lines, and hencetheir effect on particles, may also be employed. By converging ordiverging flows, particles may be combined or separated. In addition,optical peristalsis may be combined with differential effects of viscousdrag or electrical fields to produce complex and specific sets ofparameter values for finely separating materials, for example. Byopposing the trapping and other forces, the balance point of the twoforces determines whether a particle moves with the trap or the otherforce.

In one embodiment consistent with the present invention, opticalperistalsis may be implemented with a holographic system which cyclesthrough a sequence of phase patterns to implement a correspondingsequence of holographic optical trapping patterns. Such patterns may beencoded in the surface relief of reflective diffractive optical elementsmounted on the face of a prism, wherein each pattern is rotated intoplace by a motor. Likewise, transmissive diffractive optical elementsmay be placed on the perimeter of a disk and rotated to cycle throughthe patterns. Switchable phase gratings and phase holograms encoded onfilm may also be used.

For particles driven past a rectilinear array by an external bias force,such as fluid flow, where the trapping force is considerably greaterthan the external driving force, the particles are trapped. Where thebias force is greater, the particles flow past the array. Between theseextremes, the bias force exceeds the trapping force to a differingdegree for different fractions of the particles, causing the particlesto hop from trap to trap along the direction of the principal axis ofthe array. A zero net deflection may be observed where the array isrotated to 45° because: (1) positive and negative displacements occurwith equal probability; or (2) the particles become locked into the [11]direction, jumping diagonally through the array.

Particles affected to a greater degree by an array may be deflected togreater angles than the particles affected to a greater degree by thebias force. The optical gradient force exerted on particles variesroughly as a³, where a=radius. Stokes drag on the particles varies as“a”. Thus, larger particles are disproportionately affected by traparrays, while the smaller particles experience smaller deflection.Orienting the array near the angle of optimal deflection and adjustingthe intensity to place the largest particles in the hopping condition,and, hence at greater deflection than smaller particles. Differentiallydeflected particles may be collected or further fractionated byadditional arrays downstream of the first.

Some conventional techniques for fractionation achieve separation in thedirection of an applied force. However, such techniques operate onbatches of samples rather than continuously.

Other conventional techniques for microfractionation employmicrofabricated sieves consisting of a two dimensional lattice ofobstacles or barriers. For example, an asymmetric placement of barriersrectifies the Brownian motion of particles that pass through the sieve,causing the particles to follow paths that depend on the diffusioncoefficients of the particles. However, use of a microfabricatedlattices clog and are not tunable for particle size and type.

In FIG. 10, an example of sorting of particles according to the presentinvention is exemplified. Although the illustrated example exemplifieslateral deflection, optical peristalsis may be obtained in the samesystem. A representation of a video image shows light-based separationof material, in this case, tuned to separate objects based on particlesize. The flow in the upper left channel contains 1, 2.25, and 4.5 μmparticles and another flow enters from the lower left. The superimposedlines respectively indicate each of the channels' flow when the systemlaser power is off. When the laser power is turned on, light in theinteraction region (indicated by the superimposed green box), extractsthe 4.5 μm particles from the upper flow and delivers them to thelower-right channel as indicated by the superimposed white path.

6 Application in Blood Cell Sorting

a. Background

In one application consistent with the present invention, ahigh-resolution, high-throughput cell sorter by using optical trappingtechnology is implemented. The need for implementing this technology asa new basis for cell sorting is evidenced by the failure of traditionalflow cytometers to perform the high-resolution determinations of cellcharacteristics necessary in many sorting problems

b. Sorting using Holographic Optical Traps

The method of implementing high-resolution, high-throughput cell sortingof the present invention, has the following components: microfluidicdevelopment, optical-trap system development (trapping component for thefunnel system and the trap component for the separation system),high-resolution fluorescence measurement, system control (includinghologram calculation), and mechanical design.

The first component is a flow cell that has a fluid input channel,carrying the input sample, and two output channels carrying cellsseparated out of the input channel. The second component is a set oftraps that perform the “funneling” function (this “funneling function”is the equivalent of the nozzle forming the droplet flow in atraditional flow cytometer). The third component is the detection systemand, finally, the fourth component is the sorting system. FIGS. 11A-11Billustrate the relationship among these four components. Similarfunctions can be implemented with the fluid flows utilized with theapparatus 100, 200 or 300.

The essential trait allowing this proposed embodiment of the presentinvention to achieve high throughputs is its inherent capacity to runmaterial in parallel lines simultaneously and in close proximity to oneanother. For this initial implementation, a flow system with 10 inputlines 1100 each separated by 10 microns is created. This sets an overallwidth to the flow from the input reservoir of 110 microns. The outputchannels 1102, 1103 are each the same 110 micron width as the inputchannel 301 and they run parallel to the input channel 301 as is shownin FIGS. 11A and 11B. Introduced into the “output channels” 1102, 1103is a buffer solution that is fed into these channels at the same flowrate as is maintained in the input channel 1101. All three of thesechannels 1101, 1102, 1103 are designed to maintain laminar flow over theflow ranges of interest. The sorting stages discussed above with respectto the apparatus 100, 200 or 300 may also be utilized. In the sortingregion, where specific cells are transferred from the input channel 1101to one of the output channels 1102, 1103, all three flows are adjacentwith no mechanical separation between them. The laminar flows keep anymaterial in their respective flows unless a specific external force isintroduced to transfer that material from one flow channel to another.

The funneling traps 1105 act on the input cells 1106 so they both travelin well defined lines of flow and so the input cells 1106 are separatedfrom one another by a minimum distance 1106 to be set by the operator.The flow rates in the channels 1101, 1102, 1103 are set by this minimumdistance 1106, by the “update” rate of the device that is performing theseparation function, and by the overall cell processing rate desired

The funneling system is composed of a pattern of low intensity traps1105 established by a set of static holograms that are mounted in arotating wheel so that the pattern changes as a function of the rotationpattern. The most down stream funneling traps are of fixed intensity andposition, serving only to maintain the separation between the cells'lines of flow. The upstream traps 1105 are allowed to change bothintensity and position with time to act so as to disturb the flow onclumped cells and pass through individual, or un-clumped, cells.

The measurement upon which the sorting determination is made may occurin the downstream region of the funneling traps 1105 or it may occur ina region further beyond the funneling system. For this initial system,the measurement will consist of high resolution fluorescence detection.In the future, however, other active sorting criteria may beimplemented, such as scattering measurements, or passive techniques maybe employed such as those using optical deflection as outlined earlier.

The final component of the device is the separation system in which thesorting criteria is utilized to divert cells into one of the outputchannels 1102, 1103 or to allow them to remain in the flow of the inputchannel 1101. The crucial parameter for this component is thefield-of-view of the high-numerical-aperture objective lens 1104 used toimplement the array of dynamic traps 1105 driving the separation. Thewidth of this field-of-view is the same 110 microns as the individualchannels' widths. The length, however, depends upon the flow rates, thechannel depths, and the update rates of the optical device used tocontrol these traps.

Currently, one embodiment consistent with the present invention includesspatial light modulators that create phase masks which are highlyeffective in driving optical trapping systems. These devices have updaterates of 30 Hz or more. With an estimated channel depth of 10 microns,and assuming that the sperm cells should be moved in 1 micron steps, 10updates of the spatial light modulator are employed to move a cell fromthe center of the input channel 1101 to the center of either outputchannel 1102, 1103. With an update value of 30 Hz, the implementation ofthese 10 steps will occur in ⅓ second. At a flow rate of 3 mm/second,these 10 steps are implemented on a length of 1 mm in the direction offlow. The objective lens 1104 for the separation component wouldtherefore have a working area of 110 microns×1000 microns. An importantdevelopment area of this project is the design of this lens assembly.The trade-off in lens design generally is between field-of-view andnumerical aperture. That is, for a lens assembly of a particularcomplexity, a significant performance increase in one of these areaswill come with a decrease in performance in the other area. It is forthis reason that the high-performance lenses used in areas such as thehigh-resolution lithographic production of integrated-circuitelectronics are quite complex. The present invention; however, does notrequire the full performance levels of these lens assemblies.

7. Disclosure on Wide-Field Vortex Tweezing

Tweezing with a wide field of view involves microscope objective lensesthat have a relatively low numerical aperture. The ability to opticallytrap objects in the axial direction relies on focusing a light beam downin a manner that will have the largest gradients in the axial direction.This implies that a cone of light be formed with the broadest possibleradius. The radius of the cone is directly determined by the numericalaperture of the objective, i.e., high numerical aperture means a broadcone radius. This is in direct conflict with the requirements for widefield of view. This has traditionally made tweezing with a wide field ofview in the axial direction difficult. One of the major contributions tothe difficulty in axial tweezing is the radiation pressure of thefocused light beam. Especially for particles that are well matched indensity to the surrounding medium, for example polystyrene microspheres,radiation pressure may blow particles out of the trap. With a lownumerical aperture objective, it is difficult to overcome the radiationpressure with sufficient tweezing force in the axial direction. However,holographic optical traps have the ability to form exotic modes of lightwhich greatly reduce the radiation pressure of the light beam. Vortextraps, for example, have a dark center because the varying phases oflight cancel in the center of the trap. This dark center means most ofthe rays of light which travel down the center of the beam no longerexist. It is exactly these beams which harbor most of the radiationpressure of the light, so their removal greatly mitigates the difficultyin axial trapping. Other modes, e.g. donut modes; have the sameadvantage.

Manipulation (pushing, steering, sorting) of objects or cells ingeneral, is made safer by having multiple beams available. Like a bed ofnails, multiple tweezers ensure that less power is introduced at anyparticular spot in the cell. This eliminates hot spots and reduces therisk of damage. Any destructive two-photon processes benefit greatlysince the absorption is proportional to the square of the laser power.Just adding a second tweezer decreases two-photon absorption in aparticular spot by a factor of four

Finally, manipulation of even just a single cell is greatly enhanced byutilizing holographic optical trapping. A single cell may be manipulatedby a line of tweezers, which lift the cell along the perimeter on oneside. The resulting rotation allows a 360 degree view of the cell. Inaddition to the advantage for viewing of biological samples, there alsoexists the ability to orient samples stably, which has clear benefit forstudies such as scattering experiments which have a strong dependence onorientation of the sample.

8. Spinning Disk-Based Cell Sorter

The technology for using lasers to access a large number of sitesquickly already exists in the form of a spinning laser disc, CD player,or DVD player. These devices combine rotational motion of the disc withradial motion of the laser to access sites with incredibly high speeds.For example, the typical DVD player may access approximately 4 billionseparate “bits” on the disc in about two hours. Combining this spinningdisc approach with optical trapping (see FIG. 12) allows access to cellsat similar rates, and holographic optical trapping increases these ratesby factors of 100 or even higher.

FIG. 12 illustrates a spinning disc-based cell sorter in accordance withone embodiment consistent with the inventions of the second and fifthrelated applications. As shown in FIG. 12, objects or cells areintroduced at the sample intake 1200, and using an appropriate sampledelivery system 1201, the cells are provided to the sample distributiondisc 1202 which is rotated by a motor control. The imaging and trappingsystem 1203, which is connected to a control and analysis system 1204,sorts the cells and they are collected in sample chambers 1205 and 1206.

There are many mechanisms for distributing the cells over the surface ofthe disc. Fluid chambers which house individual cells, gels whichimmobilize the cells, sticky or waxy surfaces which bind the cells, oreven freezing the cells into a solid mass, are all methods that may beemployed. Once the cells are situated such that they maintain theirrelative positions, they may be appropriately measured. Optical trappingmay then be used to free either the desired or unwanted cells from thesurface or volume. In situations where sorting into more than two groupsis desired, each group may be released in a single pass, and multiplepasses may be executed.

9. Sorting of Cells and Non-Biological Material Using MeltableSubstrates

Technologies such as Fluorescence-Activated Cell Sorting (FACS),although well-established, suffer from the fact that they are serialprocessing methods. Because of the ubiquity of labeling dyes in biology,sorting on the basis of these dyes is possible. These dyes often createa difference in absorption of some wavelength or range of wavelengthsbetween dyed and undyed specimens, assuming that groups that are to besorted do not already inherently exhibit such an absorption difference.Holographic optical traps may then be used to both heat and manipulatethe specimen into a substrate which melts from the raised temperature ofthe specimen. The specimen which is embedded may then be released laterwith an increase in the bulk temperature. In addition, a faster, evenmore parallel processing method is possible in which the cells areilluminated by a broad, high power light source which processes theentire array of specimens simultaneously. The same set of methods may beapplied to non-biological samples which differ in the absorptionspectra, or may be selectively made to do so.

10. Gel-Based Sorting

Holographic optical laser traps construe a great advantage on themanipulation of objects in that they are able access and move objects inthree dimensions. As biological sorting applications become moreadvanced, larger numbers of specimens need to be sorted, often in smallamounts of time. The three-dimensional access of holographic opticaltraps means that these sorting applications may be realized. Quantifiesof cells and other specimens of biological interest which would becumbersome or impossible to sort serially or on a two-dimensionalsubstrate, may be effectively sorted.

One implementation of such three dimensional sorting relies on areversible gelation process. The cells are gelled in a network, and theneither wanted or unwanted cells are extracted from the gel usingholographic optical traps. The heat from the traps may be used to meltthe gel and provide exit pathways.

Alternatively, cells are selectively killed based on some criterion withthe holographic optical laser traps. The entire gel is then melted andthe live cells are separated from the dead. Instead of just killing, amore destructive thermal explosion may be generated, which disintegratesthe cell into much smaller components, and then sorting on the basis ofsize may be effected, grouping or connecting certain cells togetheragain.

11. Killing of Biological Specimens

A large variety of applications benefit from the ability to selectivelykill biological specimens. Removing pathogens from blood is one suchapplication. Cell sorting is another application. Cells are identified,one or more groups of cells are killed, and then the dead cells areremoved. The killing is performed by the light energy from the lasersthemselves, and do not necessarily require optical traps to perform thisfunction.

Essentially, the cells are heated or the medium around the cells areheated with the laser beam, damaging and killing the cell. Holographicoptical traps, because of they versatility and three-dimensionalcontrol, allow selective, massively parallel killing of cells.

12. Example

Using a BioRyx 200 System (Arryx, Inc., Chicago, Ill.) platelets may betweezed from whole blood. The platelets tweeze at a low laser power (0.2W) for 532 nm and they move easily in 3-D. It is preferable to use aslightly higher power for sending the platelets through automated traps,although 0.8 W is sufficient. Even in the presence of anti-coagulant,the platelets still have short strings of fibrin attached to them. Overlong periods of time, the platelets may irreversibly bind to the coverslip. Platelets are roughly 2-3 micrometers in size, and they tweezealmost as well as 2-3 micron silica at 532 nm. When the RBCs are in thesame viewing frame as the platelets, they tend to be repelled by theout-of-focus light cone, even if the RBCs are well away from the traps.However, if the red blood cells come into contact with the laser, thelaser will puncture them and often cause them to explode, depending onthe osmolarity of the medium and the laser power. Different types ofWBCs respond slightly differently to the laser tweezers. In general,WBCs are slightly repelled from them. Such differential responses tooptical traps provide a basis for separating types of cells by theirreaction to trapping beams. For example, platelets may be trapped andmoved with steered laser beams while RBCs and certain WBCs are repelledand yet other WBCs are trapped and moved to an intermediate degree.

By combining techniques described above, it may be calculated that onemay separate blood cell components at a rate of 10¹¹ platelets per 20minutes. Higher rates of sorting may be achieved by further combiningthese techniques with the laminar flow sorting of the apparatus 100, 200or 300.

The various techniques described above may be used for sorting a widevariety of matter. For sorting sperm, for example, sperm may be sortedbased upon motility or viability, such as by motile sperm moving orswimming into a selection stream, or by non-motile or nonviable spermsedimenting into a waste stream. Sperm may also be sorted into multiplechannels, each having different average motility. Sperm may also beisolated and separated from various pathogens or otherwise undesirablematerials in the semen mixture. The separation described above may alsobe utilized for washing and/or cooling processes. In addition, yields ormotile or viable sperm may be improved, for example, by manipulating thetemperature of the various flows, and the chemical content of the flows,such as by adding attractants or repellants.

FIG. 25 illustrates the results of bovine sperm viability or motilitysorting using the various embodiments of the present invention, in whicha high motility and viability sample was generated from a lowerviability and motility sample. Frozen sperm were thawed and rinsed insaline to remove glycerine, test yolk, and other materials, in order toprovide density matching to the buffer solution or saline, PEG and BSAutilized in a second flow. Alternatively, glycerine may be added to thebuffer flow). Flow rates of 0.01 to 0.1 ml/min were used, with 0.025ml/min used most commonly, in a sorter such as the apparatus 200. Spermconcentrations of approximately 5 million cells/ml with viabilities of5-60% or higher were utilized as the input flow solution. Followingsorting, the selected flow was found to have up to 80% viability, withresults anticipated to approach 90-100% viability and motility.

Various other sorter configurations also may be utilized, and improvedresults may also occur through the use of laser steering in conjunctionwith the laminar flow-based sorting. For example, increasing buffer flowspeed relative to input flow increases the width of the buffer channelin the separation region, decreasing the distance that sperm must moveto enter the buffer flow (and increasing the distance to exit the bufferflow), increasing yield in the buffer flow (as the selected flow).Increasing the waste channel flow rate may also improve yield, forcingany dead sperm in the buffer layer near the input channel to bereintroduced into the waste channel.

The sorting described above may also utilize gradients to enhancesorting efficiency, with different flows having different properties,creating gradients such as, for example, temperature gradients, velocitygradients, viscosity gradients, and diffusion gradients.

In addition to sorting, the various embodiments of the invention mayalso be utilized to change concentrations of particles or cells, forexample, such as increasing a concentration of particles in theselection stream, or diluting a concentration through an input buffersolution. Diffusion coefficients may also be manipulated, altering thediffusivity (or motility) of the objects in the various separationstreams, such as through altering temperature, chemical concentrations,fluid viscosity, fluid density, salt concentrations, use of surfactants,etc. to, for example, alter the hydrodynamic radius or surfaceattraction of an object.

As a consequence, in accordance with the present invention, theplurality of holographic optical traps, which are capable of beingindependently manipulated, can be utilized in conjunction with anapparatus 100, 200 or 300, to manipulate components or particles, suchas blood cells and other blood components, from one flow to anotherflow, as part of a separation stage. For example, components of interestin flow one may be identified and moved by the holographic optical trapsinto flow two, and thereby separated from the other components of flowone.

FIG. 6 is a flow diagram illustrating a method embodiment of the presentinvention, and provides a useful summary. Beginning with start step 600,the method provides a first flow having a plurality of components, step605, such as a plurality of blood components. A second flow is provided,step 610, and the first flow is contacted with the second flow toprovide a first separation region, step 615. A first component of theplurality of components is differentially sedimented into the secondflow, step 620, while a second component of the plurality of componentsis concurrently maintained in the first flow, step 625. The second flowhaving the first component is differentially removed from the first flowhaving the second component, step 630. When no additional separations(or stages) are to occur, step 635, the method may end, return step 680.

When an additional separation is to occur, step 635, the method proceedsto step 640, and a third flow is provided. The first flow is contactedwith the third flow to provide a second separation region, step 645.When holographic manipulation is to be utilized in the second,additional separation, step 650, the method proceeds to step 655, and aplurality of holographic traps are generated, typically using opticalwavelengths. Using the holographic traps, the second component of theplurality of components is differentially moved into the third flow,step 660. When holographic manipulation is not to be utilized in thesecond, additional separation, step 650, the method proceeds to step665, and the second component of the plurality of components isdifferentially sedimented into the third flow. Following either step 660or 665, a third component of the plurality of components is concurrentlymaintained in the first flow, step 670. The third flow having the secondcomponent is then differentially removed from the first flow having thethird component, step 675, and the method may end, return step 680.While not separately illustrated in FIG. 6, it should be understood thatthe method may continue for additional separation stages, such as athird fourth, fifth, and so on.

Additional embodiments of the invention are illustrated in FIGS. 14-24.FIGS. 14 and 15 illustrate sorting systems 1400 and 1500 employing thevarious sorting stages of the apparatus 100, 200 or 300, including theuse of reservoirs and peristaltic pumps for fluid flow. FIG. 16 is alateral view and FIG. 17 is a plan view of a high-aspect ratio flatsorter 1600. Such high-aspect ratio sorters may be utilized to provide acomparatively large laminar flow separation surface between the variousflows, providing a greater area of contact for component separationbetween the flows.

FIG. 18 is a perspective view of a three-dimensional sorting devicehaving a plurality of flat sorters (with one illustrated, such as sorter1600). FIG. 19 is a plan view of a multi-channel sorter 1700. FIG. 20 isa plan view of a sorter 1800 having a narrow waste flow region 1801.FIG. 21 is a plan view of a sorter 1900 using different flow rates forthe various channels. FIG. 22 is a plan view of a sorter 2000 havingmultiple selection channels 2001. FIG. 23 is a plan view of a sorter2100 having a constricted sorting region 2105.

FIG. 24A is a lateral view and FIG. 24B is a plan view of a multi-layerlaminar flow sorter 2200, with input channels in layer 1 (2210), layer 2providing a plurality of sorting stages (2200), and layer 3 providingoutput channels (2230). These various sorting stages may also beconnected in innumerable series and parallel connections. Numerous othervariations of such a multi-stage sorter will be readily apparent tothose of skill in the art.

Other shapes and configurations of channels may also be utilized. Forexample, a tortuous or “snake”-like channel configuration may beutilized to provide a longer interaction region in a small area orvolume. Flow control posts may also be utilized in the separationregion, to regulate the fluid flow in the channels. For example,vertical posts provide obstacles to fluid flow, effectively reducingchannel size and the Reynolds number, and improving laminar flow.

Also in summary, and by way of example, the first component of theplurality of components may be a plurality of red blood cells and aplurality of white blood cells, while the second component is aplurality of platelets. In the second separation, the plurality of whiteblood cells may be holographically separated from the plurality of redblood cells, using techniques such as holographic (optical) trapping.Holographic trapping may also be utilized to holographically remove aplurality of contaminants from the first flow, or to holographicallyseparate biological debris from the first flow. In the variousembodiments, the first flow may substantially comprise whole blood froma donor and an anticoagulant, and the second flow may substantiallycomprise plasma from the donor. The various sedimentation steps may berate zonal or isopycnic. The various flows are substantiallynon-turbulent, and may also be substantially laminar.

The fast and second separation regions each have a predetermined lengthsubstantially parallel to a direction of flow and a predetermined depthsubstantially perpendicular to the direction of flow, the predeterminedlength and predetermined depth having been determined from a firstsedimentation rate of the first component, from a second sedimentationrate of the second component, from a first flow rate of the first flow,and from a second flow rate of the second flow. The first flow and thesecond flow may have substantially the same flow rates. Alternatively,the first flow may have a first flow rate and the second flow may have asecond flow rate, in which the second flow rate is comparatively greaterthan the first flow rate.

Also in summary, the present invention further provides an apparatus forseparating a fluid mixture into constituent, non-motile components,including: (1) a first sorting channel (110 or 325) having a first inlet(120 or 315) for a first flow and a second inlet (120 or 320) for asecond flow; the first sorting channel further having a first outlet(130 or the continuous channel of FIG. 3) for the first flow and asecond outlet 130 (or 330) for the second flow, the first sortingchannel adapted to allow a first component in the first flow, of aplurality of components in the first flow, to sediment into the secondflow to form an enriched second flow and a depleted first flow, whileconcurrently maintaining a second component of the plurality ofcomponents in the first flow; (2) a second, optically transparentsorting channel (110 or 340) having a first optical inlet coupled to thefirst outlet (the continuous channel of FIG. 3) for the first flow andhaving a first optical outlet (350), the second, optically transparentsorting channel further having a second optical inlet (335) for a thirdflow and a second optical outlet for the third flow (345); and (3) aholographic optical trap system (400, 500) coupled to the second,optically transparent sorting channel, the holographic optical trapsystem adapted to generate a holographic optical trap to select and movethe second component from the first flow into the third flow.

Another apparatus or system for separating a plurality of components ina fluid comprises: an optically transparent sorting channel 100, 200 or300 having a first inlet for a first flow and a second inlet for asecond flow, the optically transparent sorting channel further having afirst outlet for the first flow and a second outlet for the second flow;and a holographic optical trap system coupled to the opticallytransparent sorting channel, the holographic optical trap system 500adapted to generate a holographic optical trap to select and move afirst component in the first flow, of a plurality of components in thefirst flow, into the second flow to form an enriched second flow and adepleted first flow, while a second component of the plurality ofcomponents is concurrently maintained in the first flow.

Lastly, another method embodiment provides for separating a plurality ofcells, comprising: providing a first flow having the plurality of cells;providing a second flow; contacting the first flow with the second flowto provide a first separation region; and differentially sedimenting afirst cell of the plurality of cells into the second flow whileconcurrently maintaining a second cell of the plurality of cells in thefirst flow. The method generally also includes differentially removingthe second flow having the first cell from the first flow having thesecond cell. The method may also provide for providing a third flow;contacting the first flow with the third flow to provide a secondseparation region; and differentially sedimenting the second cell of theplurality of cells into the third flow while concurrently maintaining athird cell of the plurality of cells in the first flow. In addition, aplurality of second cells may be holographically separated from thefirst flow, and a plurality of contaminants or biological debris may beholographically removed from the first flow.

While discussion above has focused on the sorting of blood components tocreate different blood fractions, the apparatus, methods and systems ofthe present invention may be extended to other types of particulate,biological or cellular matter which are non-motile, which are capable ofsedimenting or creaming within a fluid flow, or which are capable ofbeing manipulated optically. For example, the methodology of the presentinvention could be utilized to separate non-motile or non-viable spermcells from viable cells, by allowing the non-motile cells to sedimentfrom a first flow into a second flow. Other sorts of cell separation mayalso be performed, such as separating islet cells from other types ofpancreatic cells, or otherwise separating islet cell clusters ofdifferent sizes, through either or both flow separation or opticaltweezing (trapping). Viruses, proteins and other large molecules havingdifferent sedimentation rates may also be separated with the presentinvention. The holographic optical trapping utilized with the variousseparation stages may also be particularly useful in these other typesof cell or particle separations.

From the foregoing, it will be observed that numerous variations andmodifications may be effected without departing from the spirit andscope of the novel concept of the invention. It is to be understood thatno limitation with respect to the specific methods and apparatusillustrated herein is intended or should be inferred. It is, of course,intended to cover by the appended claims all such modifications as fallwithin the scope of the claims.

1.-71. (canceled)
 72. An apparatus to identify at least one componentfrom a plurality of components in a fluid mixture, the apparatuscomprising: a first input channel into which a first flow is introduced,said first flow which contains the fluid mixture of the plurality ofcomponents; a plurality of buffer input channels, into which additionalflows of buffer solution are introduced, said plurality of bufferchannels which are disposed on either side of said first input channel;wherein said first flow and said additional flows have a flow directionalong a length of the apparatus from one end of the apparatus to anotherend of the apparatus; a plurality of selection channels disposed at saidanother end of the apparatus, said plurality of selection channels whichare adapted to receive said additional flows enriched by selectedcomponents of the plurality of components, said selected componentswhich are selectively removed from said first flow to said additionalflows; a waste channel through which unselected components are removedfrom said first flow which is depleted of said selected components; aplurality of pumps connected respectively to a plurality of inputreservoirs, to control flow rates of said first flow and said additionalflows entering said first input channel and said plurality of bufferchannels of the apparatus, respectively; and a computer connected to theapparatus, said computer which is adapted to provide user input forcontrol of a selection of one of the plurality of components from thefluid mixture.